Extension of a protein-protein interaction surface to inactivate the function of a cellular protein

ABSTRACT

Acidic amino acid extensions to multimeric proteins, particularly nucleic acid (e.g., DNA or RNA) binding proteins, provide novel acidically modified proteins which can inhibit the function of cellular proteins, thereby regulating and controlling cell growth. The acidically modified nucleic acid binding proteins are engineered to contain a plurality of acidic amino acids appended to the proteins, generally as extensions of the multimerization or dimerization domain at the amino terminus, and can replace the basic region DNA binding domain of a DNA binding protein. The acidically extended nucleic acid binding proteins act as potent dominant negatives which were demonstrated to inhibit the activation of endogenous transactivators, such as AP1. The invention provides novel methods to create such acidically modified DNA binding proteins which can specifically and stably heterodimerize with cellular regulatory proteins and control cell growth. Suitable nucleic acid binding proteins for acidic extensions include members of transcription regulatory protein families, e.g., bZIP and HLH proteins, having characteristic leucine zipper motifs and helix-loop-helix motifs, respectively. The amino terminal extensions of the basic regions of acidically modified nucleic acid binding proteins are comprised of a sequence of amino acid residues, all or some of which are acidic in nature, and produce robust dominant negatives to the native counterpart proteins in the cell. The acidic amino terminal extension affords a unique protein-protein interaction surface and allows stable multimerization or dimerization between a native protein and the acidically extended protein, thereby controlling, via inhibition or inactivation, the functions of cellular protein products of diverse species, including plants, animals, microorganisms, and viruses.

[0001] This application is a divisional of patent application U.S. Ser.No. 09/299,495, filed Apr. 26, 1999, the disclosure of which is herebyincorporated by reference herein in its entirety, which is a divisionalof patent application U.S. Ser. No. 08/690,011, filed Jul. 31, 1996, nowU.S. Pat. No. 5,942,433, which applications claim the benefit of U.S.Provisional Application No. 60/018,496, filed May 29, 1996 and of U.S.Provisional Patent Application No. 60/001,654, filed Jul. 31, 1995.

FIELD OF THE INVENTION

[0002] The present invention relates to the production ofsequence-specific DNA binding proteins which function as eukaryotictranscription factors, i.e., transcription regulatory proteins. Theinvention more particularly relates to the generation of multimericproteins having nucleic acid (i.e., DNA or RNA) binding domains in whichthe binding domain or protein interaction surface is engineered ormodified to be acidic in nature. Such a nucleic acid binding proteinhaving an acidic multimerization domain is capable of regulating thefunction of a target nucleic acid sequence or gene to which it is bound,thereby acting as potent dominant-negative regulators of genetranscription, and cell growth and proliferation.

BACKGROUND OF THE INVENTION

[0003] Dominant-negative proteins are capable of inhibiting the bindingof nucleic acid binding proteins, i.e., DNA binding proteins, such astranscription regulatory proteins, to target DNA sequences to inactivategene function. (I. Herskowitz, 1987, Nature, 329:219-222).

[0004] The basic-region leucine zipper (“bZIP”) DNA binding proteins area family/class of nucleic acid binding proteins, which are eukaryotictranscription regulatory proteins that regulate transcription of genesby binding as dimers to specific DNA sequences. bZIP proteinscharacteristically possess two domains—a leucine zipper structuraldomain and a basic domain that is rich in basic amino acids (C. Vinsonet al., 1989, Science, 246:911-916). The two domains are separated by ashort segment known as the fork. Two bZIP proteins dimerize by forming acoiled coil region in which the leucine zipper domains dimerize. Thebasic regions then interact with the major groove of the DNA molecule ata specific DNA sequence site. The binding to DNA stabilizes the dimer.The dimerization and DNA-interaction event regulates eukaryotic genetranscription.

[0005] The leucine zipper motif is common to the primary structure of anumber of DNA binding proteins, including the yeast transcription factorGCN4, the mammalian transcription factor CCAAT/enhancer-binding proteinC/EBP, and the nuclear transforming oncogene products, Fos and Jun, andis characterized by a repeat of leucine amino acids every seven residues(i.e., a heptad repeat); the residues in this region can formamphipathic α-helices. The leucine-rich amphipathic helices interact andform a dimer complex, called a leucine zipper, at the carboxyl terminus(W. H. Landschultz et al., 1988, Science, 240:1759-1764; A. D. Baxevanisand C. R. Vinson, 1993, Curr. Op. Gen. Devel., 3:278-285), such that thedimerization region forms a coiled coil (E. K. O'Shea et al., 1989,Science, 243:538-542).

[0006] Another class of DNA binding proteins, which have similarities tothe bZIP motif, are the basic-region helix-loop-helix (“bHLH”) proteins(C. Murre et al., 1989, Cell, 56:777-783). bHLH proteins are alsocomposed of discrete domains, the structure of which allows them torecognize and interact with specific sequences of DNA. Thehelix-loop-helix region promotes dimerization through its amphipathichelices in a fashion analogous to that of the leucine zipper region ofthe bZIP proteins (R. I. Davis et al., 1990, Cell, 60:733-746; A.Voronova and D. Baltimore, 1990, Proc. Natl. Acad. Sci. USA,87:4722-4726). Nonlimiting examples of hHLH proteins are myc, max, andmad; myc and mad are known to heterodimerize.

[0007] The existence of the leucine zipper in the dimerization region ofbZIP proteins allows for a high degree of biological control through theformation of both homodimers and heterodimers. For example, heterodimersare known to form between Fos and Jun (D. Bohmann et al., 1987, Science,238:1386-1392), among members of the ATF/CREB (CRE binding protein)family (T. Hai et al., 1989, Genes Dev., 3:2083-2090), among members ofthe C/EBP family (Z. Cao et al., 1991, Genes Dev., 5:1538-1552; S. C.Williams et al., 1991, Genes Dev., 5:1553-1567; and C. Roman et al.,1990, Genes Dev., 4:1404-1415), and between members of the ATF/CREB andFos/Jun families (T. Hai and T. Curran, 1991, Proc. Natl. Acad. Sci.USA, 88:3720-3724). In general, dimerization of bZIP proteins dependsupon the ability of both of the individual carboxyl terminal α-helicesto line up in correct register with one another and to generate asymmetric coiled coil. This, in turn, places the amino terminal basicregions in a symmetric orientation, thus allowing them to interact withDNA (A. D. Baxevanis and C. R. Vinson, 1993, Curr. Op. Gen. Devel.,3:278-285). It has been shown that the ability of the helices within thecoiled coil to find the proper register with respect to one another iscontrolled inherently by the individual helices themselves, and not bythe placement of the basic region with respect to the DNA (W. Pu and K.Struhl, 1993, Nucleic Acids Research, 21:4348-4355). However, it will beappreciated that the generation of a symmetric coiled coil structure isnot a mandatory requirement for the interaction of the multimerizationor dimerization domains of various types of nucleic acid bindingproteins.

[0008] The bZIP proteins are highly conserved throughout the eukaryotickingdom and have been isolated and identified in yeast, plants, andmammals. These proteins mediate a variety of biological processes,including oncogenesis, memory, segmentation, and energy regulation (R.Boussoudan, 1994, Cell, 79:59-68; S. Cordes, and G. Barsh, 1994, Cell,79:1025-1034; S. McKnight et al., 1989, Genes Dev., 3:2021-2024; and I.Verma, 1986, Trends in Genetics, 2:93-96.). Therefore, the ability toinhibit the activity of those proteins associated with oncogenesis orabnormal cell growth and proliferation, for example, is a desirable goalin the field.

[0009] In addition, inhibition of the production or function of othercellular proteins that are detrimental, or that influence unwanted orinappropriate phenotypes, in cells, tissues, and, ultimately, the wholeorganism, is an aim for practitioners in the art.

[0010] Of the nearly 70 bZIP proteins that have been identified to date,(H. Hurst, 1994, Protein Profiles, 1:123-168), most can be categorizedinto one of five major subfamilies on the basis of their DNA recognitionproperties and amino acid sequence similarities (P. F. Johnson, 1993,Mol. Cell. Biol., 13:6919-6930). These bZIP subgroups include the AP-1,CREB/ATF, C/EBP, PAR (Proline- and Acidic amino acid Rich protein), andplant G-box proteins. The proteins in each subfamily recognize highlysimilar or identical DNA sites whose consensus sequences are 9- or10-base pair palindromes composed of two 5-base pair half-sites. Bindingsites for the various classes of bZIP proteins may differ either bytheir half-site sequences or their half-site spacing properties. AP-1proteins, such as Fos, Jun, and GCN4 (“General Control of Nitrogen andpurine metabolism factor-4”) bind to a 9-base pair pseudopalindromicsequence that can be viewed as two half-sites that overlap by a singlebase pair, while the consensus binding sites for the other four familieshave directly abutted pairs of half-sites (N. B. Haas et al., 1995, Mol.Cell. Biol., 15:1923-1932). In addition, thyrotrophic embryonic factor(TEF), a transcription factor expressed in the developing anteriorpituitary gland, and the liver-enriched albumin D box-binding protein(DBP), (C. R. Mueller et al., 1990, Cell, 61:279-291), have beenreported to constitute another class of bZIP proteins (D. W. Drolet etal., 1991, Genes Dev., 5:1739-1753).

[0011] bZIP proteins lacking the transactivation domain are naturallyoccurring dominant negatives that are generally produced by a geneticdeletion of the transactivation domain (A. Clark and K. Dougherty, 1993,Biochem J., 296:521-541; P. Descombes and U. Schibler, 1991, Cell,67:569-579; N. Foulkes et al., 1991, Cell, 64:739-749; and J. Yin etal., 1994, Cell, 79:49-58). These truncated bZIP proteins are able todimerize and bind to DNA, and if over-expressed, can act as dominantnegatives, presumably by competing with the endogenous bZIP protein forits promoter DNA binding site. Accordingly, the truncated bZIP proteinsact by mass action to occlude the normal transactivator from the DNA. Inaddition, it is possible that the deletion of the transactivation domaincould also produce a protein having increased, rather than decreased,DNA binding properties. If this were the case, then this type oftruncated and naturally occurring dominant negative would not have to beover-expressed to generate particular phenotypes (A. Braiser and A.Kumar, 1994, J. Biol. Chem., 269:10341-10351).

[0012] Needed in the art are proteins, expressed and operative in cells,having dominant-negative function to control the transcription of genesor which regulate RNA production and function in a cell. Such expressedproteins can be used for regulating abnormal cell growth in a variety ofeukaryotic organisms, including plants, animals, mammals, includinghumans, insects, microorganisms, and viruses. The present inventionprovides to the art proteins which can be modified in a particular wayto control gene regulation. The particular type of modification maycontrol gene function, for example, to inhibit abnormal or cancer cellgrowth and proliferation, to inhibit pathogenic diseases caused bymicroorganisms, particularly eukaryotic microorganisms, such as yeast,and the like, or viruses and may be used as therapeutics for treatingpathological diseases and cancer.

SUMMARY OF THE INVENTION

[0013] The present invention provides multimeric acidically modifiednucleic acid (i.e., DNA or RNA) binding proteins, such as transcriptionregulating proteins, which have been engineered to contain in theirmultimerization or protein interaction domains at least one aminoterminal acidic amino acid residue. The acidic nature of such nucleicacid binding proteins affects the binding of the proteins to otherproteins, e.g., forming heterodimers or heteromultimers, and, ultimatelythe binding of the proteins to a target DNA or RNA sequence or gene.Nucleic acid binding proteins containing an extension of acidic aminoacid residues have an extended protein interaction surface ormultimerization or dimerization interface. DNA binding proteins are aparticular example of nucleic acid binding proteins suitable for acidicmodification according to the present invention. RNA binding proteinsare also suitable for use in the invention. In accordance with theinvention, the acidic nature of the protein increases the stability ofheteromultimeric or heterodimeric complexes that are formed.

[0014] It is an object of the present invention to provide an acidicextension to a protein-protein interaction surface or dimerizationinterface to inactivate the function of a cellular protein. Inaccordance with the invention, proteins which are useful as drugs,inhibitory molecules, or growth-controlling agents or compounds areprovided which, when used both in vitro and in vivo, can inhibit theexpression and activity of cellular proteins, the effects of which canbe harmful, deleterious, and even lethal, to cell growth and survival.

[0015] It is another object of the invention to add acidic amino acidresidues to create a multimerization or dimerization surface orextension onto a multimeric complex, particularly a protein, polypeptideor peptide having basic regions that bind to nucleic acids, such as DNAor RNA. More particularly, the acidic extension appended onto themultimeric protein can replace the basic region of such proteins tocreate molecules that regulate and control cell growth.

[0016] It is another object of the invention to regulate genetranscription and expression by providing suitable dominant negativemutant molecules having an acidic phenotype that specificallyheterodimerize with native proteins to disrupt the normal action of thenative proteins in vivo, thereby causing the subsequent inactivation ofcellular gene products.

[0017] It is another object of the invention to create dominant negativetranscription regulatory proteins by extending the protein-proteininteraction surface, making it acidic in nature, to inactivate thefunction of a cellular protein by specifically and stoichiometricallydisplacing the native protein from its normal binding with DNA and byinhibiting transactivation and, ultimately, gene transcription andprotein production.

[0018] It is another object of the invention to provide improved,genetically engineered dominant negative transcription factor proteinsthat stoichiometrically inhibit the DNA binding of DNA binding proteins,such as bZIP and bHLH proteins, and structurally related types ofproteins, to limit the pleiotropic effects normally associated withnaturally occurring dominant negative over-expression. The dominantnegative mutant proteins of the invention are created by the addition ofacidic residues the N-terminus of a multimeric protein, most preferablya DNA binding protein, to produce an acidically modified protein thatcan interact with a normal cellular protein.

[0019] It is another object of the invention to provide methods andrationally-designed constructs suitable for producing and expressing thedominant negative proteins described herein for the specificinactivation of cellular gene products and for use in gene therapytechniques.

[0020] It is another object of the invention to utilize the acidicextension of the engineered DNA binding proteins to stabilize a varietyof different basic regions of proteins and to create robust dominantnegative protein members of various families of DNA binding proteins.

[0021] It is yet another object of the invention to provide transgenicanimals harboring at least one genetically engineered plasmid constructor vector containing a DNA sequence encoding a DNA binding protein thatis acidic in nature to control gene expression in a tissue specificmanner.

[0022] It is a further object of the invention to provide transgenicanimals harboring at least one genetically engineered plasmid constructor vector containing a DNA sequence encoding a DNA binding protein thatis acidic in nature which behaves as a dominant negative to the wildtype protein and provides viable phenotypes to evaluate and assess thein vivo effects of the protein. Such animals may also be used forrational drug design (i.e., with the dominant negatives considered asdrugs) and for testing and evaluating additional or supplementaltreatments, drugs, therapies, and the like, and for ameliorating oralleviating the produced transgenic phenotypes.

[0023] It is another object to provide dimeric and multimeric nucleicacid binding proteins of plant and animal origin and having acidicmultimerization domains allowing the nucleic acid binding protein tobind to a target DNA or RNA sequence, i.e., a specific gene, therebyregulating the function of the gene to which it is bound. Such proteinsare expressed, as described herein, from expression eukaryotic orprokaryotic vector constructs molecularly engineered to contain isolatedDNA sequences encoding a nucleic acid binding protein having anacidically extended dimerization or multimerization domain.

[0024] It is a further object of the invention to control the regulationof a gene through the type of acidic modification that is made to anucleic acid binding protein, thereby providing therapeutic applicationswhere the target gene or DNA or RNA sequence is present in abnormal ordiseased cells and tissues and their normal counterparts. Theengineered, acidic nucleic acid binding proteins provide tools for usein cancer therapeutics, diseases caused by eukaryotic microorganisms,for example, yeast, protozoans, algae, parasites, or by viruses, as wellas tools for drug development, rational drug design, and drug and genetherapies.

[0025] Further objects and advantages afforded by the invention will beapparent from the detailed description hereinbelow.

DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 shows thermal melts of chimeric proteins containing theCEBP leucine zipper and three bZIP basic regions derived from CEBP, VBP(Vitellogenin Binding Protein) and GBF, (“G-Box Factor”), (CEBP,VBP-CEBP, and GBF-CEBP), mixed with the dominant negative 4heptad-F. Theresults show that the 4heptad acidic extension interacts similarly withthree, different basic proteins, i.e., the ellipticity for all three is65° C.

[0027]FIG. 2 shows the protein sequence of the 4heptad extension (topline), SEQ ID NO:53, and a modified version of the 4heptad extension(new extension:4heptad) which has one amino acid change (N to L), SEQ IDNO:54, which produces a better dominant negative to the proteins Jun andCREB. Specifically, an asparagine in the a position of the first acidicheptad has been changed to a leucine, resulting in a more potentdominant negative to bZIP proteins containing a hydrophobic amino acidin the same a position, e.g., Jun, CREB, opaque, and ATF2 (“ActivatingTranscription Factor-2”). The basic region of the bZip protein Jun isshown (SEQ ID NO:55) and the basic region of the bZip protein C/EBP isshown (SEQ ID NO:56).

[0028]FIG. 3 demonstrates thermal melting curves of CREB. As shown, themelting temperature of unmodified CREB homodimerization is 47° C.(Tm=47° C.) and the melting temperature of CREB containing 4 heptadamino acid residue repeats appended at the amino terminus of the CREBprotein (4heptadCREB homodimerization) is 22° C. (Tm=22° C.). Themelting temperature of dimerization of the CREB protein with 4heptadCREBis 53° C. (Tm=53° C.). An acidically extended CREB was designed tocontain a new acidic extension (New4hepCREB) in which the asparagine waschanged to a leucine as described in FIG. 2. As shown,CREB+New4heptadCREB have a melting temperature of 69.5° C. (opencircles). This dramatic increase in thermal stability relative to theasparagine-containing acidic extension suggests that different classesof acidic extensions can be used depending on the exact bZIP protein tobe inactivated.

[0029]FIG. 4 demonstrates thermal melting curves showing the extent ofheterodimerization between the oncogenic DNA binding proteins of thebZIP class, c-Fos and c-Jun, as a consequence of engineering ormodifying the expressed c-Fos protein to contain an amino terminalextension of acidic amino acid residues. As shown in FIG. 4, the mixtureof unmodified c-Fos and c-Jun melted as 50° C. (closed circles). Anengineered c-Fos protein having the basic region deleted (0heptad-Fos)increased the melting temperature of the Fos-Jun complex to 53° C. (opensquares). An engineered c-Fos protein having the basic region replacedwith an acidic region containing four heptad repeats (4heptad-Fos)increased the melting temperature of the Fos/Jun complex to 61° C. (opencircles). The plasmid carrying DNA encoding 4heptad-Fos has beendeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, under ATCC Designation No. 97583,on May 21, 1996. An engineered c-Fos protein in which one amino acid inthe acidic region was changed from an asparagine to a leucine, asdescribed for FIG. 2, called new4heptadFos (or N4heptad-Fos), resultedin a higher melting temperature of 72° C., and more stableheterodimerization with c-Jun (closed squares). The plasmid carrying DNAencoding N4heptad-Fos has been deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, underATCC Designation No. 97584, on May 21, 1996. Also as shown in FIG. 4,homodimerization of unmodified c-Jun had a melting temperature of 29.4°C., and homodimerization of acidically modified c-Fos (having fouracidic repeats appended at the amino terminus of the c-Fos protein) hada melting temperature of 25° C.

[0030] FIGS. 5A and 5B: FIG. 5A shows that the specificity of the basiczipper region (bZIP) of the protein CEBP and the acidic extension on theN-terminus of a leucine zipper of the protein 4heptadFos is determinedby the leucine zipper for DNA binding proteins having basic and acidiczipper regions (ZIP classes of proteins). A ZIP DNA binding proteinhaving an acidic extension appended N-terminally is an acidicallymodified protein and is termed aZIP herein. As shown in FIG. 5A, mixinga bZIP CEBP protein and an aZIP 4heptadFos protein with incompatiblezippers produces no interaction, i.e., 4heptadFos does not interact withCEBP. FIG. 5B shows that mixing a bZIP VBP protein and an aZIP 3heptadF(CEBP specific) protein also produces no heterodimeric interaction. InFIGS. 5A and 5B, the solid lines represent the simple sum of the twohomodimer curves. The fact that the actual mixture gives identicalresults to the sum curve demonstrates that no heterodimers are formed.

[0031]FIG. 6 shows the results of transient transfections of human cellswith expression constructs designed to contain cytomegalovirus (CMV)promoter and DNA sequences encoding various DNA binding proteinsengineered to have acidic amino acid extensions resulting in expressedacidically modified DNA binding proteins. The constructs harbored DNAsequences encoding the cloned and isolated Fos gene having no acidicextensions (0hep-fos), or the Fos gene sequence modified to encode from1-4 acidic extensions appended thereto (i.e., 1hep-fos, 2hep-fos,3hep-fos, and 4hep-fos, respectively). Other constructs contained DNAencoding unmodified Fos (bZIP-Fos); CREB having 4 acidic extensions(4hep-CREB); VBP having 4 acidic extensions (4hep-VBP); and Jun having 4acidic extensions (4hep-Jun). In these experiments, human hepatoma cells(HepG2) were transiently transfected with up to three plasmids, e.g., achloramphenicol acetyl transferase (CAT) expression plasmid driven by asingle AP1 cis transactivation element, and different DNA bindingprotein-encoding DNAs. CMV566 AND CMV500 are plasmids containing thecytomegalovirus promoter driving the expression of DNA binding proteinsthat contain at their N-termini, either the Hemagglutinin or the FLAGepitopes, respectively (Example 4). The histogram presents the extent oftransactivation observed in the presence of different combinations ofexpressed transactivator and various acidically extended, expresseddominant-negative (DN) DNA binding proteins. In the absence of any DN,AP1 transactivation was from approximately 10 to 30 fold.Transactivation was inhibited by 1-3heptad-Fos. The results in FIG. 6show that an AP1 cis element is activated by Jun transactivation. Thisactivation is inhibited by the acidic extension on the Fos zipperprotein (aFos). The acidic extension appended to other leucine zipperproteins did not inhibit AP1 transactivation, thus indicating thespecificity of the inhibition.

[0032]FIG. 7 shows relative CAT activity in an additional cell system(Jurkat) demonstrating that an expressed Fos protein containing anacidic extension (aFos) is a better dominant negative than an expressedFos protein containing no acidic extension (bFos). Jurkat cells (a Bcell model) have a high level of endogenous Fos/Jun (AP1) activity.Cells were transiently transfected with two plasmids (1 μg), namely, areporter construct containing AP1 cis elements and a second constructcontaining the CMV500 promoter driving the expression of either atruncated form of Fos (bZipFos) or the Fos leucine zipper containing the4heptad acidic extension (aFos). AP1 activity was induced by theaddition of 12-O-tetradecanoylphorbol (TPA) which results in a 40-foldinduction of the reporter gene. The transactivation of the reporter geneoccurs because of endogenous AP1 activity. As demonstrated in FIG. 7,expression of the bZipFos protein by the bZipFos construct results indominant negative activity; however, expression of the aZipFos proteinby the aZipFos construct is more efficacious and has a more completeinhibition of AP1 activity.

[0033]FIG. 8 represents the Jurkat cell system as described in FIG. 7,with the addition that the leucine zipper (“zip”) construct alone isalso transfected into cells. Jurkat cells were transfected with twoplasmids and stimulated with TPA. The efficacy of three potentialdominant negatives containing the Fos leucine zipper (i.e., Bzip, zip,and Azip) were compared. These three dominant negatives are the Fos Bzipdomain without a transactivation domain (Bzip), the Fos leucine zipperwith no basic region (zip), and the Fos leucine zipper containing the4heptad acidic extension (Azip). The data show that expressed aFosinhibits AP1 transactivation significantly better than does an expressedFos zipper protein or an expressed bFos protein in the Jurkat cellsystem.

[0034]FIG. 9 shows the results of appending an acidic extension onto thedimerization domain of Max, a DNA binding protein of the bHLH class. The2heptadMax proteins used in the heterodimerization studies shown in FIG.9 are expressed Max proteins having acidic extensions as follows:DPDLEKEAEELEQENAELELEDSF, called 2heptadMax(783); (SEQ ID NO:1). Theplasmid carrying DNA encoding this acidically extended Max protein hasbeen deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, under Designation No.97585, on May 21, 1996. DPDLEKEAEELEQENAELEELEDSF, called2heptadMax(784); (SEQ ID NO:2). The plasmid carrying DNA encoding thisacidically extended Max protein has been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under Designation No. 97582, on May 21, 1996.DPDLEKEAEELEQENAELEEELEDSF, called 2heptadMax(785); (SEQ ID NO:3). Theplasmid carrying DNA encoding this acidically extended Max protein hasbeen deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, under Designation No.97586, on May 21, 1996.

[0035] AcidMax is an expressed Max protein having appended N-terminallyan acidic extension as follows: DPDEEEDDEEELEELEDSF; (SEQ ID NO:4). Theplasmid carrying DNA encoding this acidically extended Max protein hasbeen deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, under ATCC DesignationNo. 97581, on May 21, 1996.

[0036] 0heptadMax is an expressed Max protein having an acidic extensionas follows: DPDLEELEDSF (SEQ ID NO:5).

[0037] As can be seen from FIG. 9, an expressed Max protein having anappended polyglutamic acid sequence (i.e., AcidMax) heterodimerizedbetter with c-Myc than did the expressed 2heptadMax proteins having theabove-described acidic extensions.

[0038] FIGS. 10A-10D are schematic depictions of the bZIP protein C/EBPdimerization both with and without DNA. The relative positions of theactivation region, basic region, and leucine zipper within the proteinare shown. The NH₂ (amino) terminus of the protein is the activationdomain. The dashed line between the amino acids in the d positions ofthe leucine zipper represent a physical interaction. FIG. 10A: C/EBPdimerization. The basic region, presented as a bold line, is nothelical. FIG. 10B: C/EBP binding to DNA. The presence of DNA induces thebasic region to form an α—which increases the stability of the complexapproximately 10-fold. FIG. 10C: Heterodimerization between wild typeC/EBP and a C/EBP mutant having an F zipper which preferentiallyheterodimerizes with the C/EBP leucine zipper. FIG. 10D:Heterodimerization between C/EBP and 0heptad-F (i.e., the F zipper asshown in FIG. 10C, but having the basic region removed).

[0039] FIGS. 11A-11C demonstrate that acidic helical extension of theleucine zipper stabilizes dimerization with wild type bZIP protein. FIG.11A shows the amino acid sequences of the 4heptad acidic helicalextension, SEQ ID NO:57, and the three different basic regions of bZIPproteins that were examined, i.e., C/EBP, (SEQ ID NO:58); VBP, (SEQ IDNO:59); and GBF1, (SEQ ID NO:60). The first leucine position of thezipper, the invariant asparagine and arginine of the basic region, andthe tryptophan in the VBP bZIP protein are presented in bold-faced type.The coiled coil nomenclature of the basic region extending from theleucine zipper is indicated below with the hydrophobic a and d positionsin bold type. FIG. 11B depicts a proposed heterodimeric coiled coilstructure between the 3heptad acidic helical extension and the C/EBPbasic region; both nonequivalent sides are shown. The f position aminoacids are shown in both views. The amino acids in the 3heptad extensionthat have been changed from the parental C/EBP basic region are shown inbold type. The first leucine position of the zipper and the invariantasparagine and arginine of the basic region are stippled. The designedg⇄e′ and g⇄a′ electrostatic interactions are indicated by a black barbetween the amino acids. The right-most d amino acid shown, indicated byan arrow, is the first d position of the leucine zipper (T for C/EBP andL for 3heptad) The coiled coil heptad letter designations (a, b, c, d,e, f, g) are shown to the right. The supercoiling of the two helices isnot depicted. FIG. 11C is a coiled coil helical wheel diagram of theinteraction shown in FIG. 11B, with a view from the C-terminus towardthe N-terminus. The coiled coil sequence reads outward from the C- toN-termini around the wheel, starting at the e position following thefirst d position of the leucine zipper. Presumed electrostaticinteractions between g⇄e′ and g⇄a′ are indicated.

[0040]FIG. 12 shows the oligomerization of C/EBP determined in thepresence or absence of 3heptad-F using sedimentation equilibriumanalysis at 37° C. C/EBP alone (open squares) behaves as a singlespecies with an apparent MW=33,000 Daltons, suggesting a homodimerstructure. An equimolar mixture of C/EBP and 3heptad-F (closed circles)has an apparent MW=28,000 Daltons, indicating heterodimer formation.

[0041] FIGS. 13A and 13B: FIG. 13A shows the circular dichroism (CD)spectra of the C/EBP bZIP domain (4 μM), 0heptad-F (4 μM), and theequimolar mixture of C/EBP and the F zipper containing different lengthacidic extensions (0, 1, 2, 3, 4, ) (4 μM+4 μM). The minima at 208 and222 nm indicate α-helices. The sum of the spectra of C/EBP and0heptad-F, which would be observed if the two samples did not interact,is shown as a dotted line. The increase in the 222 nm signal for thedifferent mixtures suggests an increase in α-helical content. The noisebelow 210 nm is due to the absorbance of dithiothreitol (DTT). FIG. 13Bshows CD thermal melting curves at 222 nm of the samples shown in FIG.13A. The fitted curve through each of the data sets was used tocalculate T_(m). The mixtures were more stable than C/EBP alonesuggesting a stabilizing interaction between the two proteins. The barshows the amount of ellipticity at 222 nm generated by extending theleucine zipper one heptad on both proteins of the heterodimer.

[0042]FIGS. 14A and 14B show that acidic extension inhibits the bindingof C/EBP to DNA. FIG. 14A: 3heptad-F inhibits C/EBP DNA binding. Theleft panel shows a gel retardation assay of C/EBP binding to a specificDNA probe 28 bp in length. Protein was diluted four-fold in thesuccessive lanes. The right panel shows the same assay as the left,except that 3heptad-F was added at the same concentration as C/EBP,resulting in a total absence of C/EBP DNA binding. FIG. 14B:Displacement of VBP-C/EBP from DNA by five dominant negatives (DNs)assayed by fluorescence. Fluorescence of a single tryptophan located inthe basic region of VBP-C/EBP increased upon DNA binding (minutes 5-10).The addition of 1.1 molar equivalents of the F zipper or of the fourdifferent acidic extension caused a decrease in fluorescence. After 20minutes, 10 more molar equivalents were added, causing an additionaldisplacement of VBP-C/EBP from the DNA. 4heptad-F, shown in bold, causesthe most dramatic displacement of VBP:C/EBP. The bottom line shows thatmixing of VBP-C/EBP with 4heptad-F results in a new base line. This isnot observed for the other four mixtures which return to theVBP-C/EBP-only baseline.

[0043]FIG. 15 demonstrates that acidic extension inhibits C/EBPtransactivation. Human hepatoma cells (HepG2) were transientlytransfected with three plasmids, a chloramphenicol acetyl transferase(CAT) expression plasmid driven by a single C/EBP cis element, the C/EBPtransactivator, and different DNs. The histogram presents the extent oftransactivation observed in the presence of different combinations oftransactivator and dominant-negatives. In the absence of any DN,expressed C/EBP transactivated a single C/EBP site approximately 10fold. This transactivation was partially inhibited by a C/EBP lackingthe transactivation domain (ΔC/EBP), the heterodimerizing zipper(0heptad-F), and the acidic extension appended to the regular C/EBPzipper (3heptad-C/EBP). Transactivation was completely inhibited byexpressed 3heptad-F. The final two bars of the histogram showtransactivation of an expressed C/EBP protein containing an alternateleucine zipper, the GCN4 zipper. This protein was able to transactivatea C/EBP containing promoter and was not inhibited by 3heptad-F.

[0044]FIGS. 16A and 16B are schematic diagrams of plasmid vectors usedin the transfection studies as described. FIG. 16A shows the vectorpET3b.seq into which the DNA encoding DNA binding proteins (with andwithout acidic extensions) are inserted between the BamHI and theHindIII sites. FIG. 16B shows the vector CMV500-junbZip comprising theCMV promoter, and the FLAG and JunbZip sequences inserted between theNcoI and HindIII sites. The sequence range for this vector is from 1 to5809. Restriction enzyme cleavage sites, the number of cutting sites perenzyme, and their sequence positions are as follows: BamHI: 3 cuts atpositions 965, 1571, and 3615; EcoRI: 1 cut at position 2106; HindIII: 1cut at position 1245; NcoI: 4 cuts at positions 610, 898, 2342, and3077; NdeI: 2 cuts at positions 484 and 928; and XhoI: 2 cuts atpositions 1562 and 3628. FIG. 16C shows the vector CMV500-CREBbZipcomprising the CMV promoter, and the FLAG and CREBbZip sequencesinserted between the NcoI and HindIII sites. The sequence range for thisvector is from 1 to 5746. Restriction enzyme cleavage sites, the numberof cutting sites per enzyme, and their sequence positions are asfollows: BamHI: 3 cuts at positions 965, 1508, and 3552; EcoRI: 1 cut atposition 2043; HindIII: 1 cut at position 1182; NcoI: 4 cuts atpositions 610, 898, 2279, and 3014; NdeI: 2 cuts at positions 484 and928; and XhoI: 2 cuts at positions 1499 and 3565.

[0045]FIG. 17 depicts the isolated nucleotide sequence (SEQ ID NO:6) andthe translated amino acid sequence (SEQ ID NO:7) of a CREB proteinhaving an appended acidic extension. A 5′ BamHI restriction site and a3′ HindIII restriction site serve as sites for insertion of thenucleotide sequence into an expression vector, e.g., the pET3b.seqvector of FIG. 16A.

[0046]FIG. 18 depicts the isolated nucleotide sequence (SEQ ID NO:8) andthe translated amino acid sequence (SEQ ID NO:9) of CMV500-CREBbZIPprotein which has no appended acidic extension. A 5′ NcoI restrictionsite and a 3′ HindIII restriction site serve as sites for insertion ofthe nucleotide sequence into an expression vector, e.g., theCMV500-CREBbZip vector of FIG. 16C.

[0047]FIG. 19 depicts the isolated nucleotide sequence (SEQ ID NO:10)and the translated amino acid sequence (SEQ ID NO:11) ofCMV500-4heptadCREB (called New4hepCREB; FIG. 3) protein which has a4heptad appended acidic extension as described in FIG. 2. A 5′ NcoIrestriction site and a 3′ HindIII restriction site serve as sites forinsertion of the nucleotide sequence into an expression vector, such asa vector similar to that shown in FIG. 16C.

[0048]FIG. 20 depicts the isolated nucleotide sequence (SEQ ID NO:12)and the translated amino acid sequence (SEQ ID NO:13) encoding humanc-Fos protein, as used in the experiments described and shown in FIGS.4-6. A 5′ BamHI restriction site and a 3′ HindIII restriction site serveas sites for insertion of the nucleotide sequence into an expressionvector, such as a vector similar to that shown in FIG. 16A.

[0049]FIG. 21 depicts the isolated nucleotide sequence (SEQ ID NO:14)and the translated amino acid sequence (SEQ ID NO:15) ofCMV500-FosbZIP(MO) protein, as used in the experiments described andshown in FIG. 6. A 5′ NcoI restriction site and a 3′ HindIII restrictionsite serve as sites for insertion of the nucleotide sequence into anexpression vector, such as a vector similar to that shown in FIG. 16C.The nucleotide sequence as shown contains nucleotide sequence encodingthe FLAG epitope, nucleotide sequence encoding φ10, and the nucleotidesequence encoding FosbZIP.

[0050]FIG. 22 depicts the isolated nucleotide sequence (SEQ ID NO:16)and the translated amino acid sequence (SEQ ID NO:17; and SEQ ID NO:52)encoding the 4heptadFos protein, containing the 4heptad acidic extensionas described in FIG. 2 and as used in the experiments described andshown in FIGS. 4, 5A, 6, and 7.

[0051]FIG. 23 depicts the isolated nucleotide sequence (SEQ ID NO:18)and the translated amino acid sequence (SEQ ID NO:19) ofCMV500-4heptadFos leucine zipper protein, containing the 4heptad acidicextension as described in FIG. 2 and as used in the experimentsdescribed and shown in FIG. 6. A 5′ NcoI restriction site and a 3′HindIII restriction site serve as sites for insertion of the nucleotidesequence into an expression vector, such as a vector similar to thatshown in FIG. 16C. The sequence as shown contains sequence encoding theFLAG epitope, sequence encoding φ10, sequence encoding the 4heptadacidic extension, and sequence encoding the Fos leucine zipper.

[0052]FIG. 24 depicts the isolated nucleotide sequence (SEQ ID NO:20)and the translated amino acid sequence (SEQ ID NO:21) of CMV500-JunbZIPprotein. A 5′ NcoI restriction site and a 3′ HindIII restriction siteserve as sites for insertion of the nucleotide sequence into anexpression vector, such as the vector shown in FIG. 16B. The sequence asshown contains sequence encoding the FLAG epitope, sequence encodingφ10, sequence encoding the JunbZip protein.

[0053]FIG. 25 depicts the isolated nucleotide sequence (SEQ ID NO:22)and the translated amino acid sequence (SEQ ID NO:23) encoding AcidMax,the bHLH Max protein containing an acidic extension comprising a tractof polyglutamic acids appended to the dimerization domain of Max asshown.

[0054]FIG. 26 depicts the isolated nucleotide sequence (SEQ ID NO:24)and the translated amino acid sequence (SEQ ID NO:25) encoding2heptadMax(783), the bHLH Max protein containing two amphipathic acidicextensions, as described in FIG. 9 and in Example 6, appended to thedimerization domain of Max as shown.

[0055]FIG. 27 depicts the isolated nucleotide sequence (SEQ ID NO:26)and the translated amino acid sequence (SEQ ID NO:27) encoding2heptadMax(784), the bHLH Max protein containing two amphipathic acidicextensions, as described in FIG. 9 and in Example 6, appended to thedimerization domain of Max as shown.

[0056]FIG. 28 depicts the isolated nucleotide sequence (SEQ ID NO:28)and the translated amino acid sequence (SEQ ID NO:29) encoding2heptadMax(785), the bHLH Max protein containing two amphipathic acidicextensions, as described in FIG. 9 and in Example 6, appended to thedimerization domain of Max as shown.

[0057]FIG. 29 depicts the isolated nucleotide sequence (SEQ ID NO:30)and the translated amino acid sequence (SEQ ID NO:31) encoding murinec-Myc bHLH protein, as used in the experiments described and shown inFIGS. 4-6. Murine, rat and human c-myc sequences are highly conservedand virtually identical. A 5′ BamHI restriction site and a 3′ HindIIIrestriction site serve as sites for insertion of the nucleotide sequenceinto an expression vector, such as a vector similar to that shown inFIG. 16A.

[0058]FIG. 30 is a schematic diagram of a plasmid vector (approximately11.9 kb) which was linearized by cutting with the restriction enzymesHindIII and NotI, as indicated by the asterisks (*). The resultinglinearized plasmid DNA contained the 422 or aP2 promoter (approximately7.6 kb) contained within the HindIII-PstI site as shown. The 422 or aP2promoter is known to be associated with the regulation of the expressionof adipocyte-specific genes during adipocyte (fat cell) differentiation.The 422/aP2 gene encodes the adipose fatty acid-binding protein. The 5′flanking sequence of the 422/aP2 gene contains regulatory regions foradipose-specific expression (Bernlohr et al., 1985, J. Biol. Chem.,260:5563-5567; Spiegelman et al., 1983, J. Biol. Chem., 258:10083-10089;Cook et al., 1988, Proc. Natl. Acad. Sci. USA, 85:2949-2953). Theplasmid also contained, operably linked therewith, nucleotide sequence(approximately 0.3 kb) encoding the 3heptadC/EBP dominant negative(Flagφ10 3heptadF), in accordance with the invention as describedhereinabove, contained within the KpnI-SmaI site as shown, and afragment of approximately 1 kb which contained the polyadenylation sitefrom the early region of Simian Virus 40 (SV40) and a splice-donoracceptor sequence from the small t antigen intron from SV40 (Gorman etal., 1982, Mol Cell. Biol., 2:143-190). The insert containing the422/aP2 promoter, the Flagφ10 3heptadF C/EBP, the polyadenylation andsplice sites was cloned between the HindIII and BamHI sites of thepolylinker of the vector Bluescript KS+ (Stratagen). The linearizedplasmid was used to transfect mice for transgenic mouse production andbreeding, employing techniques routinely known in the art.

[0059]FIG. 31 shows the results of the transgenic mouse experimentsusing the plasmid DNA construct described and shown in FIG. 30. Twolitter mates resulting from successful breeding of transgenic animalsare shown: the smaller mouse near the left side of the cage exhibits thetransgenic phenotype and has the scruffy, scrawny appearance of a skinnyor thin mouse, relative to the normal-sized, non-skinny littermate onthe right, which does not carry the transgene. The thin, scruffytransgenic mouse as shown in the figure was found to possess severalcopies of the transgene as assayed by Southern Blot hybridization.

DESCRIPTION OF THE INVENTION

[0060] The present invention provides the production of novel multimericnucleic acid (i.e., DNA or RNA) binding proteins and transcriptionregulatory proteins which function as potent dominant negative proteinsto regulate gene expression and to inhibit cellular protein productionand function. Those skilled in the art will appreciate that DNA and RNAare also referred to as polynucleic acids to which the proteins of theinvention can bind. These novel proteins are produced by adding acidicamino acid residues (e.g., glutamic acid, aspartic acid) to a dimeric ormultimeric protein, e.g., bZIP or bHLH transcription regulatoryproteins, such that an engineered acidic extension, comprised of acidicamino acids, is appended onto the protein. For example, a basic regionof a multimeric DNA or RNA binding protein can be replaced with anacidic region to yield a dominant negative function. Alternatively, forexample, a plurality of acidic amino acids can be appended to a nucleicacid binding protein, preferably onto the N-terminus of the protein, toafford an acidic nature to the resulting protein. As will be appreciatedby those having skill in the art, and as exemplified and describedherein, the acidically extended nucleic acid binding proteins of theinvention are produced using conventional molecular techniques bymanipulating isolated DNA sequence encoding a nucleic acid bindingprotein such that the DNA sequence encoding the dimerization ormultimerization domain of the protein contains nucleotides which encodeone or more acidic amino acid residues. Also, DNA encoding nucleic acidbinding proteins having acidic extensions, or parts of such DNA, e.g.,DNA encoding an acidic extension, can be synthetically produced usingDNA synthesis techniques conventionally known in the art.

[0061] The proteins to which an acidic region has been extended or addedcan be derived from a variety of protein types, nonlimiting examples ofwhich include DNA or RNA binding proteins, transcription regulatoryproteins, such as the bZIP and the bHLH classes of proteins, e.g., fos,jun, opaque, DBP, ATF2, CREB, Max and Myc (e.g., N-myc, C-myc, andv-Myc). Several members of the bZIP class of transcription factors, orstructurally similar types of proteins, have functional roles inactivating the transcription of genes, the protein products of which aresubsequently expressed and function during the growth response of cells.Some of these transcription regulatory proteins are closely linked totumorigenesis, oncogenesis, and to mediating the production of proteinsinvolved in a variety of aspects of cell growth and differentiation.Such transcription factors are prime candidates for conversion intodominant negatives to control the activity of their wild typecounterparts and, ultimately, to regulate or inactivate the function ofparticular cellular protein products. It will be apparent to those inthe art that the terms wild type protein, native protein, naturallyoccurring protein and non-mutant or non-mutated protein are synonymousas used herein. It will also be understood by those in the art that thenucleic acid binding proteins of the invention may be derived fromplants, animals, including mammals and humans, microorganisms such asyeast and fungi, protozoans, algae, and parasites, as well as from RNAand DNA viruses.

[0062] More specifically, those skilled in the art are aware thatnon-mammalian eukaryotic organisms, such as plants, have nucleic acidbinding proteins, which are involved with plant physiology. GBF-1 is anexample of a plant nucleic acid binding protein of the bZIP class of DNAbinding proteins, which binds to the most divergent cis element of thebZIP proteins. Acidic extensions to plant nucleic acid binding proteinsare encompassed by the invention to modulate or regulate genetranscription. Indeed, an acidic extension appended onto GBF-1 resultedin stable heterodimerization (see Example 11 and Table 2). In otherorganisms, preferably eukaryotic and mammalian organisms, tissuespecific regulation can be effected by appending an acidic extensiononto a molecule such as a protein or polypeptide in accordance with theinvention to cause controlled regulation, in particular cell and tissuetypes, of a cellular protein that is associated with the physiology andendogenous growth of the organism.

[0063] Applications for use of the acidically extended proteins inaccordance with the invention include the growth of specific organs andtissues by selective regulation of proteins expressed during plantdevelopment. For example, storage proteins may modified to achievehealthier and more vigorously-growing plants, vegetables and crops. Asanother nonlimiting example, plant or crop growth can be augmented orotherwise modified by appending acidic extensions in accordance with theinvention onto suitable endogenous regulatory molecules to increasecarbohydrate production, for example, in potato tubers, and the like.The use of acidic extensions appended onto specific proteins,polypeptides, and cellular regulatory molecules is intended to havebroad application throughout the plant kingdom. For example, it isenvisioned that both monocotyledonous and dicotyledonous plants can beused, as well as stem and leaf vegetables (e.g., broccoli, lettuce,spinach, cabbage), fruit and seed vegetables (e.g., tomato), fiber cropsand cereals (e.g., corn, oats, wheat), and forest and ornamental crops(e.g., cotton). In addition, other uses include controlled regulation ofproteins to either enhance or decrease the growth of specific plantorgans and tissues by reducing the expression or effectiveness ofendogenous growth-associated proteins, for example, in fruits (e.g.,grapes, citrus fruits, apples, pears, apricots, and the like), or toregulate leaf, root, stem or petiole growth (e.g., cabbage, spinach,celery, beets, soybeans, sugarcane, flower stalks). Plant tissues whosegrowth and development may be regulated in accordance with the inventioncan result in the modification of various traits, such as durability,size, succulence, texture, and longevity. The control of the expressionof certain genes in accordance with the invention may reduce theexpression of growth regulatory genes or genetic control elements thatregulate gene expression to result in such nonlimiting and exemplaryuses as the dwarfing of stems for durability or enhanced mechanicalstability, genetic pruning, stunting, or the elimination of undesirableplant organs.

[0064] Examples of candidate nucleic acid binding and transcriptionregulating proteins suitable for creating dominant negatives to use asinhibitory and regulatory compounds or drugs in accordance with theinvention include any dimeric or multimeric DNA binding protein to whichan acidic extension may be appended or which can be manipulated tocontain acidic amino acid residues. Nonlimiting examples of suchproteins are the bZIP family of transcription regulatory proteins havingbasic and leucine-zipper domains, especially, proteins in the Jun andFos families, LRF-1 (liver regenerating factor 1), (J. C. Hsu et al.,1991, Proc. Natl. Acad. Sci. USA, 88:3511-3515); C/EBP, GCN4, DBP,CHOP-10 (“C/EBP Homologous Protein-10”), GBF-1; and the bHLH familyhaving basic domains and helix-loop-helix motifs, such as ID, MyoD1(“Myogenic Differentiation Factor-1”), E12 (Immunoglobulin EnhancerBinding Protein-12”), c-myc, n-myc, i-myc, max, AP-4 (“ActivatingEnhancer Binding Protein-4”), TFE3 (“Transcription Factor E3”), USF(“Upstream Stimulatory Factor”), and FIP (“Fos Interacting Protein”),(A. D. Baxevanis and C. R. Vinson, 1993, Curr. Op. Gen. Devel.,3:278-285); as well as other structurally similar proteins.

[0065] In a general sense, the invention includes the production ofacidically extended nucleic acid binding multimeric complexes, e.g.,proteins, polypeptides, or peptides, which bind to a nucleic acid, i.e.,DNA or RNA. The products are nucleic acid binding proteins having anacidic extension of amino acids in the dimerization or multimerizationdomain of the multimeric complex. The multimeric complex will usuallyhave a basic region, which may be replaced with the acidic extension tocreate molecules which are capable of controlling cell growth andproliferation when such molecules are introduced into and expressed incells. This is achieved by engineering the nucleotide sequences encodingnucleic acid binding proteins such that nucleotides encoding acidicamino acid residues are present in N-terminal extensions to the basicregion or multimerization or dimerization domains of the proteinsultimately expressed.

[0066] Dominant negatives (DN) to both bZIP and bHLH proteins containingbasic regions, with and without a leucine zipper motif, have beendeveloped and genetically engineered in accordance with the invention byaltering and replacing the normal basic domain of the wild type proteinwith one or more acidic regions. Expression constructs containing DNAsequences which produce the acidically modified DN proteins thatfunction to regulate or control cell growth (e.g. by inhibiting genetranscription or gene activation) when introduced into cells aredescribed.

[0067] In one embodiment of the invention, an acidic extension isappended onto an expressed nucleic acid binding protein. Particularlyuseful nucleic acid binding proteins for use in the invention are DNAbinding proteins. In general, one or more acidic amino acid residuescomprise the appended acidic extension. The extension can compriserepeating domains or regions, wherein each region or domain comprises asequence of amino acids, some or all of which are acidic amino acidresidues. Each domain or region comprising the appended extension cancomprise from two to about one hundred total amino acids, with eachdomain or region engineered to contain at least one, more preferably twoor more, acidic amino acids. An appended acidic extension generallycomprises two or more acidic domains or regions comprising a pluralityof amino acids appended to the DNA binding protein. The number of acidicdomains or regions comprising the acidic extension appended to theprotein, as well as the total number of amino acid residues comprisingeach domain or region, can be modified as desired or required so as toproduce an optimum dominant negative or inhibitory function. In general,an appended extension having two or more acidic regions (e.g., heptads)with at least one (e.g., two to five) acidic amino acids in each region,results in more potent dominant negative or inhibitory function. As aparticular but nonlimiting example, an acidic extension can contain fourheptad amino acid regions or domains comprising twenty-eight total aminoacid residues, with at least two amino to four amino acids in eachregion or domain being acidic (FIG. 2).

[0068] In accordance with the invention, a sequence of amino acidresidues, at least one of which, preferably two or more, is an acidicamino acid residue, can be appended onto a DNA binding protein to yieldan extended protein interaction surface or an extended dimerizationinterface that is acidic in nature. The invention encompasses an acidicextension which comprises a sequence of acidic amino acid residues, allof which are acidic, e.g., glutamic acid and/or aspartic acid. Theinvention also encompasses an acidic extension which comprises asequence of acidic amino acid residues, some of which are acidic. As ageneral guide, when all of the amino acids in the extension are notacidic, then about 1% to about 98% of the amino acids in the appendedsequence can be acidic. Thus, the appended extension can comprise asequence of amino acids which are not necessarily grouped in regions ordomains, some or all of which are acidic residues. As a simple, butnonlimiting example, the appended acidic extension can comprise fifteento twenty glutamic acid residues appended onto the multimerization ordimerization domain of the nucleic acid binding protein. Accordingly,the total number of amino acids in the sequence of amino acid residuesextended onto the protein can be of variable length, for example, fromabout two to one hundred amino acid residues, preferably from aboutthree to fifty amino acid residues, more preferably from about three tothirty amino acid residues, most preferably from about four totwenty-eight amino acid residues, some or all of which are acidic aminoacids. It is to be understood that the length of the acidic extensionmay impact upon the specificity of dimerization and DNA binding functionof the resulting DNA binding protein. In addition, it will beappreciated by those in the art that acidic extensions of varying length(i.e., long and short extensions) can be appended onto a protein toincrease or decrease the potency of the resulting dominant negativeprotein; i.e., to make a stronger or more toxic “drug” comprising theacidically-extended DNA binding proteins of the invention, the number ofacidic amino acids in the extension can be increased as required. Thoseskilled in the art will appreciate that the number of acidic amino acidscan be changed to change the strength of an acidically extended proteinto suit the needs of the practitioner. For example, a dominant negativeDNA binding protein having a short acidic extension (e.g., three tothirteen amino acids) can be created, as well as a dominant negative DNAbinding protein having a long acidic extension (e.g., twenty to onehundred amino acids). In general, a longer acidic extension may beexpected to provide increase potency, toxicity, or stability to thedominant negative DNA binding protein to which it is appended. Theability to change the strength of the drug afforded by the acidicallyextended DNA binding proteins of the invention by increasing the numberof acidic amino acids comprising the extension allows the resultingproteins to have differential strength as regulators and controllers ofcell growth and proliferation.

[0069] In another embodiment, the acidically extended DNA bindingprotein is an expressed bZIP transcription regulatory protein having abasic region and a leucine zipper. In accordance with the invention, theprotein is engineered to contain a designed acidic amino acid sequencewhich extends N-terminally from the leucine zipper region of theexpressed bZIP protein. The extension of amino acids can comprise fromtwo to one hundred amino acid residues, preferably from about three tofifty amino acid residues, more preferably from about three to thirtyamino acid residues, some or all of which are acidic residues, therebymaking the appended amino acid extension acidic in nature.Alternatively, the acidic extension can comprise a sequence of aminoacids, all of which are acidic in nature, e.g., glutamic acid, in whichthe acidic amino acid sequence comprises from about two to eighty aminoacids, preferably from about three to fifty amino acids, and morepreferably about five to thirty amino acids. It is to be understood thatall of the amino acid residues comprising the acidic extension of thebZIP protein can be acidic amino acids or that at least one amino acidresidue in each of the amino acid regions comprising an appendedextension is an acidic amino acid (e.g., at least one amino acid residueis an acidic residue in each of four heptad repeats comprising theextension, FIG. 2). More frequently, two or more amino acid residueswithin a given region of an appended extension of the bZIP protein areacidic (FIG. 2; FIG. 11A)

[0070] In another embodiment, the extension of amino acids can beamphipathic. As described above, the extension can also be designed tooccur in one or more repeating domains or regions. Preferred are acidicextensions comprising one or more amino acid domains or regions, whereineach domain or region of amino acid residues comprises at least oneacidic amino acid residues, preferably two or more acidic residues,among the amino acids comprising each region. For example, an acidicextension containing four repeating regions with each region comprisinga sequence of seven amino acids comprised of acidic amino acid residuesis a 4heptad repeat appended onto the protein in accordance with theinvention.

[0071] As described, the acidic amino acid sequence can extend for oneor more heptad repeats from the N-terminus of the protein and can havean N-terminal cap, which may be comprised of three glycine residues(e.g., DP-GGG, (SEQ ID NO:61)). As described herein (Example 1F), theN-terminal cap may also be comprised of other sequences, namely, DP-(aspartic acid and proline alone); DP-D (aspartic acid and proline andaspartic acid); DP-EE (SEQ ID NO:62), (aspartic acid and proline and twoglutamic acid residues), and DP-DEEE (SEQ ID NO:63), (aspartic acid andproline and three glutamic acid residues) to replace the DP-GGG (SEQ IDNO:61) cap.

[0072] In another embodiment of the invention, the acidically extendedDNA binding protein is an expressed transcription regulatory bHLHprotein having a basic region and a helix-loop-helix structure. Inaccordance with the invention, the bHLH protein is engineered to containa designed acidic amino acid sequence which extends N-terminally fromthe multimerization or dimerization domain of the protein. In theseproteins the N-terminal DNA binding amino acids are deleted and replacedwith the acidic extension. The acidic extension comprises repeatingdomains or regions comprised of a sequence of amino acids having atleast one acidic amino acid in each repeating region. Alternatively, theacidic extension comprises a sequence of amino acids, some or all ofwhich are acidic, e.g., glutamic and/or aspartic acid. Such an acidicextensions can comprise from two to eighty amino acids, preferably fromthree to fifty amino acids, and more preferably four to thirty aminoacids. It is also to be understood that an acidic extension can simplycomprise a sequence of amino acids appended onto the N-terminalmultimerization or dimerization domain of a nucleic acid bindingprotein, wherein some or all of the amino acid residues in the appendedextension are acidic. As but one example, an acidic extension appendedto a bHLH protein is comprised of fifteen repeating glutamic acidresidues. In another example, as described for the bHLH protein, Max(FIG. 9 and Example 6), it was found that an expression constructresulting in the expression of a Max protein having a stretch ofpolyglutamic amino acid residues appended N-terminally thereto resultedin superior heterodimerization with c-myc protein. Plasmid vectorsharboring the DNA sequence encoding acidically modified Max proteinswere created using the N-terminal DPD amino acid sequence, which is aBamHI cloning site (Example 6).

[0073] The designed acidic region appended onto the protein, e.g., aleucine zipper protein, functioned as a dominant negative totranscription factors comprised of basic regions and leucine zipperregions; basic regions and helix-loop-helix structures, and other DNAbinding proteins that are capable of dimerization and/or of binding to atarget DNA sequence or gene and regulating or controlling the functionof the DNA target sequence or gene to which the DNA binding proteins arebound. In general, specificity is derived from the dimerization domainin the dominant negatives. It will be appreciated that the production ofnucleic acid binding proteins which are acidic in nature is applicableto protein-protein interacting systems other than those particularlydescribed and exemplified herein. As but one nonlimiting example, thebHLH family of transcription factors are considered to be goodcandidates for extending the dimerization motif, because the basicregion is an extension of the dimerization region, in a manner that issimilar to that of the bZIP protein family.

[0074] In another embodiment, the present inventors have designedconstructs which contained and expressed isolated DNA encoding a bZIPleucine zipper-containing protein (called the F zipper orheterodimerizing zipper) that preferentially heterodimerized with thewild type bZIP transcription factor C/EBP, a heat-stable DNA-bindingprotein present in rat liver (W. H. Landschultz et al., 1988, GenesDev., 2:786-800; C. R. Vinson et al., 1993, Genes Dev., 7:1047-1058).The F-zipper was created by placing charged amino acids in the e and gpositions of the coiled coil structure, thereby resulting in thepreferential formation of heterodimers over homodimers, as described inC. R. Vinson et al., 1993, Ibid.. Interestingly, the heterodimer formedby the complexation of native C/EBP and the F zipper constructed withouta basic region (i.e., C/EBP:0heptad-F) was more stable than the wildtype or native C/EBP homodimer in the absence of DNA. However, theheterodimer was not as stable as the native C/EBP homodimer bound toDNA, and thus, an equimolar amount of the heterodimerizing F zipperwithout an acidic amphipathic extension (0heptad-F) could not displacenative C/EBP from its binding to DNA. Thus, the earlier-produced Fzipper was not considered to be a good or useful dominant negative atstoichiometric concentrations.

[0075] In accordance with the present invention, the creation of novelmultimeric nucleic acid binding proteins containing an acidic extensionallows the extension to stretch the protein-protein multimerization ordimerization interface or surface. A heterodimer complex ultimatelyformed can be stabilized by over one hundred fold (i.e., 2.5 kcal/mol).For proteins having basic regions, such as bZIP and bHLH proteins,acidic extension allows the extension to increase the protein-proteininteraction interface into the basic region of the protein. The acidicextension also can increase the stability of interaction of aheterodimeric complex between the engineered protein containing theacidic extension and the native protein. The increase in heterodimerstability makes the dominant negatives of the invention effectivestoichiometric competitors of the wild type or native protein products.

[0076] In accordance with the invention and as a particular nonlimitingexample, the stability of the heterodimer formed between the wild typeC/EBP protein and the F zipper protein was increased by appending adesigned three-heptad-long amphipathic α- to the N-terminus of the Fzipper (abbreviated 3heptad-F herein), (FIGS. 11A-C). In this way, theacidic extension could electrostatically mimic DNA, thus providing theC/EBP basic region with an alternative interaction surface. In theparticular case of C/EBP, the extension of the C/EBP leucine zipper intothe basic region would create new a and d positions containinghydrophobic amino acids. For other proteins, such as the bHLH proteinsand the like, the acidic extension provides an alternative or extendedinteraction surface which ultimately controls the extent of binding of aprotein to a target DNA sequence (i.e., a gene).

[0077] For bZIP proteins, the amino acid sequence of the acidicextension forming a protein interaction surface for binding with thebasic region of C/EBP is graphically presented from both a side and anend view (FIGS. 11B-11C). The N-terminal extension was designed toprevent unfavorable stearic or electrostatic interactions with the basicregion, and to form attractive electrostatic and hydrophobicinteractions that would result in the formation of an appropriateinteraction surface. Commercially-available CPK molecular models wereused to aid in the molecular design. Leucine was placed in all the dpositions of the extension to drive potential hydrophobic interactions.Because the native C/EBP basic region contains two asparagines in the aposition, asparagine was placed in the acidic extension to createfavorable a⇄a′ interactions between the asparagines of the basic regionand the acidic extension, as reported for GCN4 leucine zipper structures(T. Ellenberger et al., 1992, Cell, 71:1223-1237). In the second andfourth a positions of the native C/EBP basic region is an arginine; thearginine in the second position is conserved in all bZIP proteins. Inaccordance with the invention, an alanine was placed in the opposing aposition of the acidic extension to avoid possible stearic clashes. Aglutamic acid was put in the g position to create an attractive g⇄a′electrostatic interaction, to parallel the type of interaction thatoccurs in the zipper of the c-Fos:c-Jun heterodimer (M. Glover and S.Harrison, 1995, Nature, 373:257-261). In addition, glutamic acid wasplaced in the e and g positions to create favorable g⇄a′ interactions.Additional glutamic acids were included in the b and c positions to helpelectrostatically neutralize the heterodimeric structure. The resultingprotein comprising the three heptad N-terminal extension and the Fzipper is referred to as 3heptad-F. The hyphen designates the junctionbetween the basic region and the leucine zipper. For heterodimers, thenames of the individual proteins comprising the heterodimer areseparated by a colon, e.g., C/EBP:3heptad-F.

[0078] Also in accordance with the invention, acidic extensions wereappended onto the dimerization domain of expressed bHLH proteins, e.g.,Max, and heterodimerization with expressed c-myc protein was observed(FIG. 9, Example 6). Increased heterodimerization stability was foundwhen the acidic extension was appended onto the bHLH protein in any ofthree different orientations, thereby showing the universal nature ofthe acidic extension to extend protein-protein interaction interfaces ofthe nucleic acid binding domains of nucleic acid binding proteins,particularly DNA binding proteins.

[0079] In accordance with the invention, it was found that the additionof one or more heptads of acidic amino acid sequences onto theN-terminus of a leucine zipper protein stabilized heterodimer formation,as determined by circular dichroism (CD) spectra and thermal meltingassays as described herein. This acidic amino acid extension provided anextended protein interaction surface with the basic region of the wildtype or native protein containing the leucine zipper and allowed thecreation of a dominant negative to the native protein thatstoichiometrically displaced the native protein from DNA and inhibitedtransactivation by the native protein.

[0080] It will be appreciated by those in the art that at least one(1heptad) and preferably two to four or more heptads, i.e., 2heptad,3heptad, 4heptad, and the like (a maximum number can be routinely andempirically determined by those having skill in the art) can be appendedto the N-terminal region of appropriate DNA binding proteins to achieveoptimal heterodimer binding stability of the proteins produced inaccordance with the present invention. However, it is also noted thatlonger acidic extensions may lead to nonspecificity as a result of anincreased interaction with more of the same or similar nucleic acidbinding proteins. Such nonspecificity may be alleviated by expressingless protein and/or by employing fewer acidic sequence extensions. Acombination of these strategies will allow for the generation of adominant negative with minimal nonspecific or pleiotropic effects.

[0081] In accordance with the invention, plasmid constructs or vectorsare provided for the production and expression of the dominant negativenucleic acid binding proteins described. These nucleic acid bindingproteins can be expressed, if desired, in a variety of expressionsystems, particularly eukaryotic and mammalian systems, e.g., yeast,insect, human, hamster, mouse, rat, and the like, using reagents andmethods known to those in the art. The constructs for use in both invitro and in vivo systems are designed to contain at least one promoter,an enhancer sequence (optional, for mammalian expression systems), andother sequences as necessary or required for proper transcription andregulation of gene expression (e.g., transcriptional initiation andtermination sequences, origin of replication sites, polyadenylationsequences). As will be appreciated by those skilled in the art, theselection of the appropriate vector and plasmid components for propertranscription, expression, and isolation of proteins produced ineukaryotic and prokaryotic expression systems is known and routinelydetermined and practiced by those having skill in the art.

[0082] Ultimately, the constructs containing the nucleic acid sequencescoding for the dominant negative nucleic acid binding proteins of theinvention are introduced into cell types of interest, having theappropriate milieu for transcription of the gene(s) whose transcriptionregulatory proteins and products are to be inhibited or negativelyregulated by the dominant negatives of the invention. The constructs canbe designed to contain the appropriate and necessary DNA elements forexpression of the dominant negative protein in a given cell type, ifdesired.

[0083] For example, the expression of a dominant negative nucleic acidbinding protein (e.g., DNA binding protein) having an acidic amino acidextension can be placed under the control of promoters such as viralpromoters, e.g., cytomegalovirus (CMV), Rous sarcoma virus (RSV),phosphoglycerol kinase (PGK), thymidine kinase (TK), or the α-actinpromoter. Further, a regulated promoter, such as the glucocorticoidresponse element (GRE) of mouse mammary tumor virus (MMTV), would conferinducibility by glucocorticoids (V. Chandler et al., 1983, Cell,33:489-499). Alternatively, tissue-specific promoters or regulatoryelements can be used (G. Swift et al., 1984, Cell, 38:639-646),non-limiting examples of which include the following: the N-CAM promoter(specific for brain and central nervous system); the PIT-1 promoter(pituitary specific transcription factor); the crystalline promoter(specific for regulating protein expression in the lens of the eye); thekeratin promoter (specific for regulating protein expression in theskin); the albumin promoter (liver-specific); the alpha- or beta-globinpromoters (specific for red blood cells); the Ig enhancer (specific forB lymphocytes); the T cell receptor α- or β-promoters (specific for Tcells); the insulin promoter (specific for pancreatic cells); thegastrin promoter (specific for cells of the stomach); the cardiac actinpromoter (specific for heart); the tropomyosin promoter (specific forskeletal muscle); and the lactalbumin promoter and the whey acidicprotein (WAP) promoter (A. C. Andres et al., 1987, Proc. Natl. Acad.Sci. USA, 84:1299-1303; C. W. Pittius et al., 1988, Proc. Natl. Acad.Sci. USA, 85:5874-5878), (specific for expression in breast tissue).

[0084] Regulated and tissue specific promoters allow the expression ofthe mutant dominant negative phenotype to be conditional, in general,thereby being exhibited if and when expression of the gene is induced.This would allow the propagation of dominant negative nucleic acidbinding proteins of the invention, in the absence of expression of thedominant negative phenotype, so that the inducible dominant negativescan be used in a manner similar to that of temperature sensitivemutations. In addition, since the modification made to DNA bindingproteins are dominant and the phenotype is exhibited in the presence offunctional wild type genes, the consequences of inactivating thefunction of genes present in multiple copies can be assessed withouthaving to inactivate each copy of the gene (I. Herskowitz, 1987, Nature,329:219-222).

[0085] As mentioned above, those skilled in the art will appreciate thata variety of promoters, enhancers, and genes are suitable for use in theconstructs of the invention, and that the constructs will contain thenecessary initiation, termination, and control sequences for propertranscription and processing of the gene of interest when the constructis introduced into a cell. The constructs may be introduced into cellsby a variety of gene transfer methods known to those skilled in the art,for example, conventional gene transfection methods, such as calciumphosphate co-precipitation, liposomal transfection, microinjection,electroporation, and infection or viral transduction. In addition, it isenvisioned that the invention can encompass all or a portion of a viralsequence-containing vector, such as those described in U.S. Pat. No.5,112,767 to P. Roy-Burman and D. A. Spodick, for targeted delivery ofgenes to specific tissues. The choice of the method is within thecompetence of the skilled practitioner in the art. It will be apparentto those skilled in the art that one or more constructs carrying DNAsequences for expression in cells can be transfected into the cells suchthat expression products are subsequently produced in and/or obtainedfrom the cells.

[0086] In the constructs of the invention, the nucleic acid sequencesencoding nucleic acid binding proteins with acidic extensions can beobtained and isolated from natural sources or by synthetic means. Forexample, using conventionally known techniques in molecular biology, theappropriate DNA sequences can be excised and isolated and purified fromgenomic clones or from cDNA clones generated from the diverse speciesknown to express nucleic acid binding proteins and the like; thenucleotide sequences can be genetically engineered to encode amino acidresidues comprising the acidic extensions as described, and theresulting nucleic acid can then be ligated with at least one of theappropriate or desired segments of DNA sequence to construct a plasmidor vector, comprising, for example, a promoter, an enhancer, terminatorsite, and polyadenylation site. Alternatively, the nucleic acidsequences can be synthesized for use in the constructs according to allor part of the sequences provided in the sequence ID numbers herein, andin the accompanying figures, by conventional techniques of DNAsynthesis, such as the phosphite triester chemistry method (for example,see U.S. Pat. No. 4,415,732 to Caruthers et al.; and Sinha, N. D. etal., 1984, Nucl. Acids Res., 12:4539-4557).

[0087] In another aspect of the invention, the acidically extendeddominant negatives constructed in accordance with the invention aretested for their in vitro and in vivo functions in inactivating therelevant family of transcription factors in an in vitro or in an in vivomilieu. With regard to in vitro studies, the ability of expressednucleic acid binding proteins produced in accordance with the inventionto inhibit cellular proteins that are associated with cell growth can beassayed using the transient transfection and transformation techniquesas described. Such techniques and assays can also be used to screen theengineered, acidically extended nucleic acid binding proteins of theinvention for use as candidate anti-cancer or disease drugs and intherapeutic applications.

[0088] With regard to in vivo studies, constructs as described for thetransient transfection experiments (Examples 1F and 8C) are used toconstruct transgenic animals, e.g., mice, as described in Example 13.The plasmid constructs carrying the CMV promoter to drive expression ofthe DNA sequence coding for the DNA binding proteins, e.g., bZIP andbHLH proteins (e.g., 4heptadFos, 4heptadCREB, 2heptadMax, and 3heptad-Fproteins), are introduced into animals. Thereafter, the effects of theexpression of the acidically extended dominant negatives are monitoredin animals harboring the DN transgenes. The animals are assayed for thepresence of the transgene and for the expression of the DN protein incells. Alternatively, DNA sequences encoding the acidically extendeddominant negative proteins of the invention are constructed in a vectorcontaining a tissue specific promoter to target the expression of thisheterologous gene to specific tissues of the transgenic animals, alongwith the other components required for a complete vector construct.

[0089] Another aspect of the invention provides tissue specificconstructs which will target those tissues in which the acidicextension-containing nucleic acid binding proteins are to be expressed.Such constructs are designed to contain tissue-specific promoters suchas those enumerated hereinabove, and a DNA sequence encoding a DNAbinding protein having acidic extensions, e.g., the 4heptadFos,4heptadCREB, 3heptadMax, or other transcription factors, the expressionof which will be driven by the tissue-specific promoter, and theappropriate initiation and termination sites for transcription, andpolyadenylation sites, if necessary, for expression in a given cell andtissue type. Thus, the invention allows the selective expression and thedetermination of the function of DNA binding protein family members,such as those of the bZIP and bHLH families, in individual tissues bymeans of the production of transgenic organisms and via genetic therapyapproaches known and used in the art.

[0090] For gene therapy applications of the present invention,constructs designed to carry the acidically extended nucleic acidbinding proteins, e.g. DNA binding transcription regulatory proteins,RNA binding proteins, and the like, as described herein can supply thedescribed DN protein inactivation function to a variety of cell types,including stem cells, differentiated cells, germ cells, and oncogenic ortransformed cells. Supplying such an inactivating or inhibitory functionby the dominant negatives of the invention can lead to and/or cause thesuppression of neoplastic growth in a variety of cell types, especiallythose cells which have been growth-altered, for example, as aconsequence of the interaction and activity of Fos-Jun dimerization(Examples 2 and 4 and FIGS. 1 and 4) or Myc-Max dimerization (Examples 6and 7 and FIG. 9). In addition, because of the divergent biologicalroots of the DNA binding proteins, the DN constructs afforded by theinvention are applicable to the areas of human biology, non-humanmammalian biology, insect biology, and plant biology. In the lattercase, the dominant negatives can modulate the expression of valuablegene products related to plant stress, anti-fungal agents, insecticides,and pesticides.

[0091] The dominant negatives, or a dominant negative-functioning partor fragment thereof, in accordance with the invention, can be introducedinto a cell in a vector such that the DNA segment remainsextrachromosomal and will be expressed by the cell from theextrachromosomal location in the cell. Vectors for the introduction ofgenes and DNA segments suitable for both recombination andextrachromosomal maintenance are known in the art. Cells transformed ortransfected with the DN-containing constructs of the invention can beused as model systems to study cancer or tumor remission.

[0092] Gene transfer systems known in the art may be useful in thepractice of the invention. Both viral and non-viral methods aresuitable. Numerous viruses have been used as gene transfer vectors,including papovaviruses, e.g., SV40, adenovirus, vaccinia virus,adeno-associated virus, herpes viruses, including Herpes Simplex Virus(HSV) and Epstein Barr Virus (EBV), and retroviruses of avian, murine,and human origin. As is appreciated by those in the art, most human genetherapy protocols have been based on disabled murine retroviruses.Recombinant retroviral DNA can also be employed with amphotropicpackaging cell lines capable of producing high titer stocks ofhelper-free recombinant retroviruses (e.g., R. Cone and R. Mulligan,1984, Proc. Natl. Acad. Sci. USA, 81:6349).

[0093] Receptor-mediated gene transfer methods allow targeting of theDNA in the construct directly to particular tissues. This isaccomplished by the conjugation of DNA (frequently in the form ofcovalently-closed supercoiled plasmid) to a protein ligand viapoly-lysine. The appropriate or suitable ligands are selected on thebasis of the presence of the corresponding ligand receptors on the cellsurface of the target cell or tissue type. These ligand-DNA conjugatescan be injected directly into the blood, if desired, and are directed tothe target tissue where receptor binding and DNA-protein complexinternalization occur. Co-infection with adenovirus to disrupt endosomefunction can be used to overcome the problem of intracellulardestruction of DNA.

[0094] Nonviral gene transfer methods known in the art include chemicaltechniques, such as calcium phosphate co-precipitation, direct DNAuptake and receptor-mediated DNA transfer, and mechanical means, such asmicroinjection and membrane fusion-mediated liposomal transfer. Inaddition, viral-mediated gene transfer can be combined with direct invivo gene transfer using liposomes, thereby allowing the delivery or theviral vectors to tumor cells, for example, and not to surroundingnon-proliferating cells. The retroviral vector producer cell line can beinjected directly into specific cell types, e.g., tumors, to provide acontinuous source of viral particles, such as has been approved for usein patients afflicted with inoperable brain tumors.

[0095] An approach that combines biological and physical gene transfermethods utilizes plasmid DNA of any size combined with apolylysine-conjugated antibody specifically reactive with the adenovirushexon protein. The resulting complex is bound to an adenovirus vector.The trimolecular complex is then used to infect cells. The adenovirusvector allows efficient binding to the cell, internalization, anddegradation of the endosome before the coupled DNA can be damaged.

[0096] The described constructs may be administered in the form of apharmaceutical preparation or composition containing a pharmaceuticallyacceptable carrier and a physiological excipient, in which preparationthe vector may be a viral vector construct, or the like, to target thecells, tissues, or organs of the recipient organism of interest,including human and non-human mammals. The composition may be formed bydispersing the components in a suitable pharmaceutically-acceptableliquid or solution such as sterile physiological saline or otherinjectable aqueous liquids. The amounts of the components to be used insuch compositions may be routinely determined by those having skill inthe art. The compositions may be administered by parenteral routes ofinjection, including subcutaneous, intravenous, intramuscular, andintrasternal. Other modes of administration include intranasal,intrathecal, intracutaneous, percutaneous, enteral, and sublingual. Forinjectable administration, the composition is in sterile solution orsuspension or may be emulsified in pharmaceutically- andphysiologically-acceptable aqueous or oleaginous vehicles, which maycontain preservatives, stabilizers, and material for rendering thesolution or suspension isotonic with body fluids (i.e. blood) of therecipient. Excipients suitable for use are water, phosphate bufferedsaline, pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose,glycerol, dilute ethanol, and the like, and mixtures thereof.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids, which may be usedeither on their own or as admixtures.

[0097] It is also envisioned that the acidically modified nucleic acidbinding proteins of the invention can be used to screen compounds usingthe expressed acidically extended nucleic acid binding proteins or afunctional binding fragment thereof in a variety of drug screeningmethods. For example, an engineered and expressed DNA binding protein orfragment employed in such a test may either be free in solution, affixedto a solid support, or displayed on a cell surface. One drug screeningtechnique utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant polynucleotides expressing the protein orfragment, preferably in competitive binding assays. Such cells, eitherin viable or fixed form, can be used in standard binding assays. Forexample, the formation of complexes between a nucleic acid bindingprotein or fragment and the agent being tested may be quantified, or thedegree to which the formation of a complex between a nucleic acidbinding protein or fragment and a known ligand is interfered with by theagent being tested can be examined.

[0098] Thus, the present invention provides methods of screening fordrugs comprising contacting such an agent with an acidically modifiednucleic acid binding protein of the invention or fragment thereof andassaying (i) for the presence of a complex between the agent and thenucleic acid binding protein or fragment, or (ii) for the presence of acomplex between the nucleic acid binding protein or fragment and aligand, by methods well known in the art. In such competitive bindingassays, the nucleic acid binding protein or fragment is typicallylabeled. Free nucleic acid binding protein or a fragment is separatedfrom that present in a protein:protein complex, and the amount of free(i.e., uncomplexed) label serves as a measure of the binding of theagent being tested to the nucleic acid binding protein or of itsinterference with the nucleic acid binding protein:ligand binding,respectively.

[0099] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to the DNAbinding proteins and is described in detail in Geysen, European PatentApplication 84/03564, published on Sep. 13, 1984. In brief, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with the DNA binding protein and washed.After washing, bound DNA binding protein is then detected by methodswell known in the art. Alternatively, purified DNA binding protein canbe coated directly onto plates for use in the aforementioned drugscreening techniques. However, non-neutralizing antibodies to thepolypeptide can be used to capture antibodies to immobilize the DNAbinding protein on the solid phase.

[0100] The invention also contemplates the use of competitive drugscreening assays in which antibodies capable of specifically binding theDNA binding protein compete with a test compound for binding to the DNAbinding protein or fragments thereof. In this manner, the antibodies canbe used to detect the presence of any peptide which shares one or moreantigenic determinants with the DNA binding protein.

[0101] Rational drug design is also envisioned as a use for the presentinvention. In general, the goal of rational drug design is to producestructural analogs of biologically active polypeptides and proteins ofinterest or of small molecules with which they interact (e.g., agonists,antagonists, inhibitors) in order to fashion drugs which are, forexample, more active or stable forms of the protein or polypeptide, orwhich, for example, enhance or interfere with the function of a proteinor polypeptide in vivo. See, e.g., Hodgson, 1991. In one approach, thethree-dimensional structure of a protein of interest (e.g., anacidically extended DNA binding protein of the invention) is firstdetermined by x-ray crystallography, by computer modelling or mosttypically, by a combination of approaches. Less often, usefulinformation regarding the structure of a polypeptide may be gained bymodeling based on the structure of homologous proteins. The productionand design of nucleic acid binding proteins having acidic amino acidextensions can lead to the expression, production and detection ofproducts having an increased affinity for binding to cognate cellularproteins than do the normal, unmodified cellular counterparts. Uponcomplexation with its naturally occurring cognate cellular nucleic acid(e.g., DNA) binding protein, an acidically extended nucleic acid bindingprotein of the invention can inhibit or prevent the function of itscognate protein and ultimately affect cellular gene transcription andexpression. Thus, the products designed and created in accordance withthe invention can yield more potent dominant negatives of wild type ornaturally occurring proteins.

[0102] Accordingly, drugs may be designed which have, e.g., improved DNAbinding function, activity or stability or which act as more potentdimerization molecules, or as inhibitors, agonists, antagonists, etc. ofDNA protein binding activity. By virtue of molecularly synthesizing theDNA binding protein sequences, sufficient amounts of the proteins may bemade available to perform such analytical studies as X-raycrystallography. In addition, DNA sequence of the DNA binding proteinwill allow computer modeling techniques to be employed in place of or inaddition to x-ray crystallography.

[0103] Active nucleic acid binding protein molecules can be introducedinto cells by microinjection or by use of liposomes, for example.Alternatively, some functioning molecules may be taken up by cells,actively or by diffusion. Extracellular application of the gene productof the acidically extended DNA binding proteins of the invention may besufficient to affect tumor growth, provided that the gene productsbecome localized in the nucleus. The supply of molecules with theactivity of an acidically extended DNA binding protein should lead topartial reversal of the neoplastic state. Other molecules withacidically extended nucleic acid binding protein activity (for example,peptides or functional portions thereof) may also be used to effect sucha reversal. Modified polypeptides having substantially similar functionare also used for peptide therapy.

EXAMPLES

[0104] The examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the inventionin any way.

Example 1

[0105] Materials and Methods

[0106] A. Nucleic Acids, Plasmids, and Proteins

[0107] Plasmid constructs harboring DNA encoding the nucleic acidbinding proteins, both with and without acidic extensions, for cellexpression were produced using routine methods, protocols and reagentsin the art. Constructs for the expression of the 4heptad-F zipperprotein were built to contain the cytomegalovirus (CMV) promoter, theFLAG epitope (Invitrogen), and the nucleic acid sequence shown in SEQ IDNO:32. The DNA sequence depicted by SEQ ID NO:32 contains an N-terminalleader sequence (described below in SEQ ID NO:33), three glycine (GGG)residues (nucleotides 43-51), which, when deleted, produce a moreeffective dominant negative, as described herein (see Example 1F), the4heptad sequence depicted in SEQ ID NO:38, and the F-zipper DNA sequenceembraced by SEQ ID NO:34.

[0108] The nucleic acid sequence of SEQ ID NO:32, and its encoded aminoacid sequence (SEQ ID NO:64) are as follows: 1ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG M   A   S   M   T   G   G   Q   Q   M   G   RGAT CCT GGC GGT GGC CTG GAA CAA CGT GCT GAG GAA D   P   G   G   G   L   E   Q   R   A   E   ECTG GCC CGT GAA AAC GAA GAG CTG GAA AAA GAG GCC L   A   R   E   N   E   E   L   E   K   E   AGAA GAG CTG GAG CAG GAA AAC GCT GAA CTC GAG CAG E   E   L   E   Q   E   N   A   E   L   E   QGAA GTG TTG GAG TTG GAA AGT CGT AAT GAC CGC CTG E   V   L   E   L   E   S   R   N   D   R   LCGC AAG GAA GTG GAA CAG CTG GAG CGT GAA CTG GAC R   K   E   V   E   Q   L   E   R   E   L   DACG CTG CGG GGT ATC TTC CGC CAG CTG CCT GAG AGC T   L   R   G   I   F   R   Q   L   P   E   S                                              296TCC TTG GTC AAG GC CATGGGCAACTGCGCGTGAGGCGAATTCAA  S   L   V   K   A

[0109] The protein sequences expressed from the constructs transformedinto E. coli, produced in vivo, and isolated and purified as describedherein are listed below. In each protein, the initiator methionine isprocessed off in E. coli. The conventional one-letter symbols forabbreviating amino acid residues are used herein and are as follows: A(alanine); C (cysteine); D (aspartic acid); E (glutamic acid); F(phenylalanine); G (glycine); H (histidine); I (isoleucine); K (lysine);L (leucine); M (methionine); N (asparagine); P (proline); Q (glutamine);R (arginine); S (serine); T (threonine); V (valine); W (tryptophan); andY (tyrosine).

[0110] All proteins, except for C/EBP and GBF-C/EBP, have a 13 aminoacid N-terminal leader sequence as follows: ASMTGGQQMGRDP- (SEQ IDNO:33).

[0111] The F-zipper amino acid sequence is:GGGTQQEVLELESRNDRLRKEVEQLERELDTLRGIFRQLPESSLVKAMGNCA (SEQ ID NO:34).

[0112] The amino acid sequences of the multiheptad-containing zippersare provided below. The L in the penultimate position in the heptadsequences is the first “L” position of the F leucine zipper. Thoseskilled in the art will be aware that the nucleic acid sequences, i.e.,the triplet codons, corresponding to the various amino acid sequencespresented herein and for use in the presently claimed invention can bedetermined in a conventional manner in accordance with the genetic code.1heptad-F GGGLEQENAELE (SEQ ID NO:35); 2heptad-F GGGLEKEAEELEQENAELE(SEQ ID NO:36); 3heptad-F GGGLARENEELEKEAEELEQENAELE (SEQ ID NO:37);4heptad-F GGGLEQRAEELARENEELEKEAEELEQENAELE (SEQ ID NO:38); 3heptad-F(cap)GGGLARNNIAVRKSRDKAKQRNVELE (SEQ ID NO:39); 3heptad-F(cap,`a`d)`GGGLARNNIALRKSADKLKQRNVELE (SEQ ID NO:40); 3heptad-F(cap,`e`g)`GGGLARENIAVEKERDKAEQENVELE (SEQ ID NO:41); 3heptad-F(cap,`b`c)`GGGLARNNEEVRKSREEAKQRNAELE (SEQ ID NO:42).

[0113] Three bZIP domains described and utilized herein are shown in1)-3) below. Unless otherwise noted, the nucleic acid sequencescorresponding to the protein sequences of the chimeric bZIP proteins arethose reported in the referenced publications.

[0114] 1) The chimeric sequence of GCN4-C/EBP protein is depicted withthe GCN4 sequence in italics:SEYQPSLFALNPMGFSPLDGSKSTNENVSASTSTAKPMVGQLIFDKFIKTEEDP (SEQ ID NO:43).GKAKKSVDKNSNEYRVRRERNNIAVRKSRDKAKQRNVETQQKVLELTSDNDRLRKRVEQLSRELDTLRGIFRQLPESSLVKAMGNCA

[0115] Cloning of GCN4 and its corresponding nucleic acid sequence havebeen described by A. G. Hinnebusch, 1984, Proc. Natl. Acad. Sci. USA,81:6442. The isolation and nucleic acid sequence of the gene encodingC/EBP are described in W. H. Landschulz et al., 1988, Genes Dev.,2:786-800.

[0116] 2) The polypeptide sequence of the GBF-C/EBP chimeric protein:PVKDERELKRQKRKQSNRESARRSRLRNEAECEQTQQKVLELTSDNDRLRKRVEQLSRELDTLRGIFRQLPESSLVKAMGNCA (SEQ ID NO:44). Cloning of the Arabidopsisthaliana-derived bZIP protein, GBF, and its corresponding nucleic acidsequence have been described in U. Schindler et al., 1992, The EMBO J.,11:1261-1273.

[0117] 3) The polypeptide sequence of the VBP-C/EBP chimeric protein:ASMTGGQQMGRDPLEEKVFVPDEQKDEKYWTRRKKNNVAAKRSRDARRLKENQTQQKVLELTSDNDRLRKRVEQLSRELDTLRGIFRQLPESSLVKAMGNCA (SEQ ID NO:45).Cloning of the chicken vitellogenin gene-binding protein VBP and itscorresponding nucleic acid sequence have been described by S. lyer etal., 1991, Mol. Cell. Biol., 11 :4863-4875.

[0118] B. Protein Expression and Purification

[0119] Proteins with and without acidic extensions were expressed in E.coli and isolated and purified from culture supernatants. Moreparticularly, proteins were synthesized in E. coli using the phage T7expression system (F. Studier and B. Moffatt, 1986, J. Mol. Biol.,189:113-130. Bacterial cultures (about 400 mL) were induced with 1 mMisopropyl-b-D-thiogalactopyranoside) at an optical density of 0.6 at 600nm for about 2 hours. Cells were then recovered by centrifugation,resuspended in 6 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA,1 mM benzamidine, 1 mM dithiothreitol (DTT), and 0.2 mMphenylmethylsulfonyl fluoride (PMSF)), frozen, thawed, and gentlybrought to 1 M KCl by the addition of 2 mL of 4 M KCl. The sample wascentrifuged at 25,000 rpm in a Beckman T42 rotor, and the supernatantwas removed and isolated. The isolated supernatant was then heated to65° C. for 10 minutes and centrifuged, and the supernatant was isolated.The proteins that were capable of binding DNA were diluted to 100 mM KCland purified over a heparin-agarose column as described by D. Krylov etal., 1994, EMBO J., 13:1849-1861, and were subsequently purified over aRainin HPLC system. The proteins lacking DNA binding domains werepurified over a Mono Q Sepharose column and eluted with 200 mM, 400 mM,and 600 mM KCl. The fractions enriched in the proteins were thenpurified on a Rainin HPLC system using a C18 column chromatographed from0% to 100% acetonitrile in 0.1% trifluoroacetic acid. The molarconcentrations were calculated using the molar extinction at 230 nm ofamino acids (300/residue). The contribution of tyrosine (4980) andtryptophan (6818) to the absorbance at 230 nm were included if necessary(C. Cantor and P. Schimmel, 1980, Biophysical Chemistry. W. H. Freemanand Co., New York; G. Fasman, 1976, CRC Handbook of Biochemistry andMolecular Biology, 3rd Edition. CRC Press).

[0120] C. Analytical Ultracentrifugation

[0121] Analytical ultracentrifugation was performed as described in D.Krylov et al., 1994, EMBO J., 13:2849-2861, except that the proteinabsorbance was monitored at 260 nm. Protein samples at threeconcentrations were centrifuged at 24,000 rpm until equilibrium and datawere collected after 48 hours. The C/EBP sample (MW=16,104), whichcontains one tyrosine, absorbed at a wavelength of 260 nm, while3heptad-F (MW=9,847), which does not contain any aromatic amino acids,does not absorb at 260 nm. This absorption difference allowed themonitoring of the oligomerization state of C/EBP in the presence orabsence of 3heptad-F.

[0122] D. Circular Dichroism (CD)

[0123] CD analyses were performed as described in Materials and Methodsof D. Krylov et al., 1994, EMBO J., 13:2849-2861. T_(m) values werecalculated as described in D, Krylov et al., 1994, Ibid, converted toK_(d(25)) and ΔG(25) using a ΔC_(p) of −0.96 kcal/mol° C.⁻¹ calculatedfrom a T_(m) versus ΔH plot for all of the proteins as described herein.All thermal melts were reversible. Table 2 shows the stability of themixtures of heterodimerizing zipper (F) with C/EBP under different saltconditions. TABLE 1 Homodimer with C/EBP Protein T_(m)(ΔG)k_(d)T_(m)(ΔG)k_(d) C/EBP (L) 45.8(−10.3)3e−8 C/EBP-F (L) 52.2(−11.1)8e−9C/EBP (H) 49.5(−10.9)1e−8 C/EBP-F (H) 28(−7.7)2e−6 53.6(−11.2)7e−90heptad-F (H) 30.0(−8.0)2e−6 55.4(−11.8)3e−9

[0124] Table 1 presents the melting temperature (T_(m), ° C.), ΔG andk_(d) at 25° C. for CD thermal melts of a variety of proteins either ashomodimers or an equimolar mixture of two proteins. If the mixture had ahigher melting temperature than either homodimer alone, it was inferredthat the mixture sample was composed of heterodimers. The (L) denotes‘low’ salt conditions (50 mM KCl, 25 mM Tris pH 8.0, 0.5 mM EDTA, 1 mMDTT) and the (H) denotes regular salt conditions (12.5 mM potassiumphosphate, pH 7.4, 150 mM KCl, 0.25 mM EDTA, and 0.5 mM DTT). The latterconditions were used in Table 2. “0heptad-F” represents the F zipperwith the basic region deleted. The RMS error in AG is 0.2 kcal/mol and0.5 kcal/mol for homo- and heterodimers respectively.

[0125] E. Tryptophan Fluorescence

[0126] Fluorescence of a tryptophan in the VBP-C/EBP basic region wasmonitored using an Aminco SLM8000 photon counting spectrofluorimeter inslow kinetics mode 25° C., with excitation and emission at 296 and 342nm, respectively. Samples contained the same salt conditions used asdescribed for the CD measurements. Samples containing 10⁻⁶ M VBP:C/EBPmonomer were mixed with a DNA probe, 28 base pairs in length andcontaining the C/EBP binding site (0.5×10⁻⁶ M of duplex), to which asequence the VBP basic region binds specifically (N. B. Haas et al.,1995, Mol. Cell. Biol., 15:1923-1932).

[0127] Haas, et al. 1995) The sequence of the probe is 5′GTCAGTCAGATTGCGCAATATCGGTCAG 3′ (SEQ ID NO:46). The consensus C/EBP DNAbinding site is shown underlined and in bold.

[0128] The K_(d)'s of homo- and heterodimers calculated from the CDthermal melts provided a determination of the fraction of VBP-C/EBPdisplaced from DNA when the DN was added in the fluorescence equilibriumexperiments. For example, the VBP-C/EBP DNA binding constant wascalculated by determining how effective the DN was at displacingVBP-C/EBP from DNA using the following equation:

[DNA_(bound)]/[DNA_(free)]=(K_(hetero)/K_(DNA))²[Heterodimer]²/{K_(DN)[DN]},

[0129] wherein K_(hetero) is the heterodimer dissociation constant,

[0130] K_(DN) is the DN dissociation constant, both measured from CDthermal melts, and K_(DNA) is the DNA binding constant of atranscription factor.

[0131] The VBP-C/EBP DNA binding constant was calculated as 10⁻¹⁰ whendata for 0heptad-F in 11 molar excess is used, or 1heptad-F, 2-heptad-For 3heptad-F in 1.1 molar excess is used. The consistency of thecalculated DNA binding constants suggests that the ΔG's for theheterodimers as determined by CD thermal melts are valuable indicatorsof efficacy.

[0132] F. Transient Transfections

[0133] The C/EBP family of transactivator plasmids (pMEX-C/EBPα,pMEX-C/EBPβ, pMEX-C/EBPδ) containing the MSV promoter used herein wereas described by S. Williams et al., 1991, Genes and Devel., 5:1553-1567.These plasmids were modified to include a FLAG epitope (Invitrogen) atthe N-terminus. The C/EBPα-GCN4 chimeras contained the leucine zipperfrom the bZIP protein GCN4. The CAT reporter plasmid contained aconsensus C/EBP binding site (i.e., ATTGCGCAAT, SEQ ID NO:47) in frontof the minimal promoter. All the DNs were cloned into pRc/CMV(Invitrogen) modified to contain the N-terminal FLAG epitope with anuclear localization sequence (MDYKDDDDK-KKRK, SEQ ID NO:48). C/EBPα wasalso cloned into pRc/CMV and gave results similar to those obtainedusing pMEX-C/EBPα for both the transactivation and inhibition assays.Similar results were obtained for plasmids modified with the FLAGepitope and without the FLAG epitope.

[0134] In these experiments, 10 μg of reporter plasmid, 0.3 μg oftransactivator, and 5 μg of DN were added to each transienttransfection. 3heptad-F with the DPD cap totally inhibited C/EBPtransactivation in a 1:1 molar ratio. Sheared salmon sperm DNA was addedto bring the DNA concentrations to 20 μg. Calcium phosphatetransfections were performed following the manufacturer's instructions(Gibco).

[0135] Western blotting of cellular extracts of generated from thetransient transfections indicated that 3heptad-F containing a FLAGepitope placed 5′ or N-terminal of the acidic extension was not seen onthe blot, even though the construct completely inhibited C/EBPtransactivation. A possible explanation was that the three glycineN-terminal cap used to stop the acidic extension was aproteolysis-sensitive protein sequence. Consequently, four additionalsequences were employed as N-terminal caps, namely, DP-, DP-D, DP-EE(SEQ ID NO:62), and DP-DEEE (SEQ ID NO:63), replacing DP-GGG (SEQ IDNO:61)), (J. Richardson and D. Richardson, 1988, Science,240:1648-1652). Their thermal stability as heterodimers with C/EBP wasmonitored, and their ability to be detected using a Western blot of cellextracts obtained from the transient transfections was determined. Allof the sequences were detected in Western blots. Interestingly, the DP-and DP-D caps were three-fold more stable than the DP-GGG (SEQ ID NO:61)cap.

[0136] It was found by the present inventors that transactivation of thebZIP protein C/EBP was not totally inhibited either by theheterodimerizing F zipper (i.e., 0heptad-F) or by a three heptad acidicextension appended onto the C/EBP leucine zipper (i.e., 3heptad-C/EBP).Notwithstanding, the 3heptad-C/EBP did inhibit to a measurable degree.Changes in both the leucine zipper and the acidic extension generated aprotein (3heptad-F) that formed a sufficiently stable heterodimer withC/EBP to inhibit competitively both C/EBP DNA binding andtransactivation. However, it will be appreciated that for other proteinsin the bZIP family and in other structurally similar families ofproteins, the acidic extension alone may contribute enough stabilizingenergy to create a dominant negative without the additional stabilityconferred by the heterodimerizing zipper. This will depend on therelative stabilizing effect of the DNA binding for particular bZIPproteins. Based on the present invention, acidic extensions can bedesigned that dimerize even more strongly with the basic region, thusallowing the creation of a dominant negative to any bZIP protein bysimply appending an acidic extension onto the N-terminus of any bZIPleucine zipper.

Example 2

[0137] Acidic Extensions Increase Heterodimerization of the bZIP Classof Proteins

[0138] Experiments were conducted to determine the effect of acidicextensions on DNA binding proteins of the bZIP class, c-Fos, CREB, andCEBP. c-Fos and CREB proteins were designed to contain an acidic 4heptad repeat (FIGS. 3 and 4). An acidically extended CREB was designedto contain a new acidic extension (New4hepCREB) in which the asparaginewas changed to a leucine as described in FIG. 3. As shown,CREB+New4heptadCREB had a melting temperature of 69.5° C. (opencircles). This dramatic increase in thermal stability relative to theasparagine-containing acidic extension suggests that different classesof acidic extensions can be used to append to the N-terminaldimerization domain of bZIP proteins, depending on the exact bZIPprotein to be inactivated.

[0139] In FIG. 4, an engineered c-Fos protein having the basic regiondeleted, called 0heptad-Fos, increased the melting temperature of theFos/Jun complex to 53° C. (open squares). An engineered c-Fos proteinhaving the basic region replaced with an acidic region (containing fouracidic repeats, called “4heptadFos”) increased the melting temperatureof the Fos/Jun complex to 61° C. (open circles). An engineered c-Fosprotein in which one amino acid in the acidic region was changed from anasparagine to a leucine, as described for FIG. 2, called new4heptadFos,resulted in a higher melting temperature of 72° C., and more stableheterodimerization with c-Jun (closed squares). Also as shown in FIG. 4,homodimerization of unmodified c-Jun had a melting temperature of 29.4°C., and homodimerization of acidically modified c-Fos (having fouracidic repeats appended at the amino terminus of the c-Fos protein) hada melting temperature of 25° C. Also in FIG. 4, the Kd(37° C.) for thec-Jun homodimer was 20 μM; 0.2 μM for the c-Jun/c-Fos heterodimer; and 6nM for the c-Jun/4heptadFos heterodimer.

Example 3

[0140] Specificity of a bZIP and the Acidic Extension is Determined bythe Zipper

[0141] The specificity of the basic region of a bZIP protein and theacidic extension on the N-terminus of a leucine zipper (i.e., calledaZIP) was found to be determined by the leucine zipper. As shown in FIG.5A, mixing a bZIP CEBP protein and an aZIP 4heptadFos protein withincompatible zippers produces no interaction, i.e., 4heptadFos does notinteract with CEBP. FIG. 5B shows that mixing a bZIP VBP protein and anaZIP 3heptadF-(CEBP specific) protein also produces no heterodimericinteraction. In FIGS. 5A and 5B, the solid lines is the simple sum ofthe two homodimer curves. The fact that the actual mixture givesidentical results demonstrates that no heterodimers are formed.

Example 4

[0142] Transactivation by an AP1 cis Element is Inhibited by AcidicExtension Appended onto a DNA Binding Protein

[0143] AP1 activity was demonstrated to be inhibited by several acidicextensions appended onto the bZIP DNA binding protein c-Fos in transienttransfection experiments using human hepatoma cells (HepG2) andAP1-dependent CAT reporter expression constructs and measuring CATactivity (FIG. 6). HepG2 cells were transfected with up to three plasmidconstructs for the expression of c-Fos proteins. The three plasmidswere: a construct for expression of the Jun transactivator, a reporterconstruct containing an AP1 cis element driving CAT expression, and aconstruct containing DNA encoding the dominant negative and appropriateexpression and regulatory sequences. CMV566 AND CMV500 are plasmidscontaining the cytomegalovirus promoter driving the expression of DNAbinding proteins that contain at their N-termini, either theHemagglutinin (YPYDVPDYA) (SEQ ID NO:49) or the FLAG (DYKDDDDK) (SEQ IDNO:50) epitopes, respectively. c-Fos proteins were designed to containno acidic heptad repeats appended N-terminally from the basic region(0hep-Fos), one acidic heptad repeat appended N-terminally from thebasic region (1 hep-Fos), two acidic heptad repeats appendedN-terminally from the basic region (2hep-Fos), three acidic heptadrepeats appended N-terminally from the basic region (3hep-Fos), or fouracidic heptad repeats appended N-terminally from the basic region(4hep-Fos). Transfections were carried out using either 3 or 15 μg ofplasmid containing CMV566 or CMV500 and DNA sequence encoding bZIPhaving N-terminal extensions as shown in FIG. 6. The results of theseexperiments show that AP1 transactivation by expressed c-Jun protein isinhibited by expressed c-Fos protein having appended acidic extensions.The results also demonstrate that the inhibition of AP1 transactivationis specific, since acidic extensions appended onto other bZIP proteins(i.e., CREB, VBP, or Jun) did not inhibit AP1 transactivation.

Example 5

[0144] A bZIP Protein Containing an Acidic Extension (aZIP) is a BetterInhibitor of AP1 Activation than a bZIP Protein Containing No AcidicExtension

[0145] Experiments similar to those described in Example 4 wereperformed in an additional transient cell system using Jurkat cellsextension (bFos). Jurkat cells (a B cell model) have a high level ofendogenous Fos/Jun (AP1) activity. Cells were transiently transfectedwith two plasmids (1 μg each), namely, a reporter construct containingAP1 cis elements and a construct containing the CMV500 promoter drivingthe expression of either a truncated form of Fos (bZipFos) or the Fosleucine zipper containing the 4heptad acidic extension (aFos). AP1activity was induced by the addition of 12-O-tetradecanoylphorbol (TPA)which resulted in a 40-fold induction of the gene. The transactivationof the reporter gene occurred because of endogenous activity. Asdemonstrated in FIGS. 7 and 8, expression of the bZipFos protein encodedby a bZipFos DNA sequence construct resulted in dominant negativeactivity; however, expression of aZipFos protein encoded by an aZipFosconstruct was more efficacious and showed a significantly high level ofinhibition of AP1 activity. In these experiments, the aZIP protein c-Foswas designed to contain 4 acidic heptad repeats appended N-terminally(4heptad-Fos). The inhibitory activity of the expressed aZIP protein wascompared with that of a c-Fos protein that was not engineered to containan acidic extension (bZIP).

Example 6

[0146] Acidic Extensions Appended to DNA Binding Proteins ProvideCompounds for the Control and Regulation of Gene Function

[0147] An acidic extension was appended onto the dimerization domain ofa DNA binding protein of the bHLH class (FIG. 9). Replacing the basicregion of an expressed bHLH protein Max having an amphipathic acidicextension (i.e., 2heptadMax) increased heterodimerization of Max withc-Myc, irrespective of the orientation of the acidic extension asappended to the HLH protein. The 2heptadMax proteins used in theheterodimerization studies shown in FIG. 9 are expressed Max proteinshaving amphipathic acidic extensions as follows:DPDLEKEAEELEQENAELELEDSF, called 2heptadMax(783), (SEQ ID NO;1);DPDLEKEAEELEQENAELEELEDSF, called 2heptadMax(784), (SEQ ID NO:2); andDPDLEKEAEELEQENAELEEELEDSF, called 2heptadMax(785), (SEQ ID NO:3).

[0148] AcidMax is an expressed Max protein having appended N-terminallyan acidic extension as follows: DPDEEEDDEEELEELEDSF (SEQ ID NO:4).0heptadMax is an expressed Max protein having the sequence DPDLEELEDSF(SEQ ID NO:5) as its acidic extension. 0heptadMax is the dimerizationdomain of Max without an acidic extension. Because of gene cloning, thisprotein as expressed has the short acidic extension DPDLEELEDSF (SEQ IDNO:5). The increase in the dimerization with c-Myc by the addition ofthe acidic extension on Max shows the utility of the approach of theinvention.

[0149] In addition, the expressed AcidMax protein having a polyglutamicacid sequence heterodimerized with c-Myc better than did Max proteincontaining any of the three amphipathic extensions. In FIG. 9, the Kd(M)for the acidically modified and unmodified HLH proteins is as follows:Homodimer Heterodimer with c-Myc Kd (M) Kd (M) bHLH MAX 6 × 10⁻⁶ 3 ×10⁻⁸  0heptadMax 1 × 10⁻⁷ 6 × 10⁻¹⁰ 2heptadMax 4 × 10⁻⁸ 6 × 10⁻¹²AcidMax 3 × 10⁻⁸ 2 × 10⁻¹²

[0150] In the plasmid vectors designed to carry the Max DNA sequence,the “DPD” amino acid sequence is a Bam cloning site. ImmediatelyN-terminal of the BamHI cloning site DPD, the protein constructs containa 13 amino acid sequence that is from the φ10 phage (ASMTGGQQMGR) (SEQID NO:51). The eukaryotic expression vectors can contain an additionalN-terminal protein sequence 5′ of the φ10 protein sequence. Thisadditional N-terminal protein sequence is an epitope used to monitorexpression in mammalian cells. Its sequence is YPYDVPDYA (SEQ ID NO:49).

[0151] The last amino acid of the Max extensions is a phenylalanine (F)in 1 of the dimerization domain of Max. This amino acid is conserved inmost bHLH proteins, thus providing a convenient landmark in the Maxprotein structure from which to know where the designed acidic sequenceswere appended onto the dimerization domain of Max. In the experimentsdescribed in this Example, an amphipathic acidic protein sequence wasadded onto the N-terminus of the Max dimerization domain. The linkerbetween the acidic extension and the dimerization domain containeddifferent numbers of glutamic acids. This difference in the length ofthe linker had the effect of rotating the acidic amphipathic sequencerelative to the dimerization domain.

[0152] These results demonstrate the universal nature of the acidicextension for allowing a DNA binding protein from different classesultimately to control and regulate gene function.

Example 7

[0153] Effects on Cell Growth and Transformation Following StableTransfection of Cells with Constructs Harboring DNA Binding ProteinsContaining Appended Acidic Extensions

[0154] Cell transfection assays were performed in which cells (e.g.,C3H10T1/2) were plated and transfected with 50 ng DNA per plate. Thefoci assay used in these experiments was as described by S. Min et al.,1993, Oncogene, 8:2691-2701. Two experiments with the potential DNs wereconducted. First, cells were transformed with the constructs asdescribed and were plated in conventional tissue culture plastic dishes(e.g., Petri dishes). These assays examined the effects that theconstructs containing DNA encoding acidically extended DNs had on cellviability. It was found that cell viability was not affected by theconstructs. Next, the same cells were transfected with constructsharboring Max DN DNA (described in Example 6) in conjunction withplasmids harboring Ras and/or Myc-encoding DNA, and the ability of thecells to grow in a contact-inhibited independent fashion was examined.The cells were plated in plastic dishes at high concentrations and theirability to overcome contact inhibited growth was evaluated. When the MaxDNs of the invention (e.g., acidically extended Max) were co-transfectedwith Ras (10 ng) and/or Myc (300 ng), the action of the DN was clearlyobserved. 0heptadMax (1 μg) inhibited foci formation from 73 colonies to34 colonies, while 2heptadMax decreased the number of colonies down tozero. A polyacidic Max construct is likely to inhibit foci formation inthese assays as well as, or better than, the inhibition activity of2heptadMax. The ras construct used in the experiments contains a rasgene which contains a mutation which keeps it constitutively active asdescribed in E. Taparowsky et al, 1982, Nature, 300:762-765. The rasgene is driven by its normal promoter. The myc construct is a regularmyc gene driven by the RSV LTR. After 3 days, plates were fixed and werestained after 14 days. Also, after 14 days, G418 resistant colonies werequantified and the results are shown below. Total Number Number of FociPlasmid Construct of Colonies Per Plate pCMV 566 509 127 pCMV 566 Max549 137 0heptad Max 510 128 2heptad783 Max 336  92 2heptad784 Max 524131 2heptad785 Max 517 129

[0155] Number of Foci Plasmid Construct Per Plate Ras 22 Ras + Myc 73Ras + Myc + pCMV566Max 52 Ras + Myc + 0heptadMax 34 Ras + Myc +2heptad784Max  0

[0156] As shown below, the same types of experiments performed using theFos constructs of the invention showed an inhibition of foci formation.In contrast to Max, the Fos expression constructs were able to inhibitboth colony formation and foci formation in these assays. In the colonyassay, results are also shown for a control plasmid construct(designated pKOneo) carrying only the neomycin gene for the selection ofcells carrying this gene and expressing its product; the control plasmidresulted in 127 colonies. As known by those in the art, the neo gene isa conventional marker gene, built into constructs containing otherexpressible genes and used as a selection marker for expression of theneo gene as determined by resistance of the cells to neomycin followingtransformation or transfection of cells with a plasmid construct asdescribed. Focus Assay: Relative Focus Plasmid Construct Formation Ras(200 ng) 1 Ras (200 ng) + 0.38 Oheptad-Fos (600 ng) Ras (200 ng) + 0.294heptad-Fos (600 ng)

[0157] Colony Assay: Colonies Per Plasmid Construct Plate pKONeo (200ng) 127  0heptad-Fos (200 ng) 121  4heptad-Fos (200 ng) 87 Ras (200ng) + 61 0heptad-Fos (600 ng) Ras (200 ng) + 49 4heptad-Fos (600 ng)

Example 8

[0158] Competition Assays

[0159] Three types of competition experiments demonstrated that theacidic extension created a robust DN to the bZIP protein C/EBP'sactivity. Two biochemical assays addressed DNA binding (i.e., gel shiftand fluorescence assays) and one biological assay addressedtransactivation function (i.e., transient transfection assay).

[0160] A. Gel Shifts

[0161] Gel shift experiments were undertaken to examine if 3heptad-Finhibited the binding of C/EBP to its cognate DNA sequence. Gel shiftdata indicated that C/EBP bound to DNA with a K_(d)=10⁻⁹ M (Z. Cao etal., 1991, Genes Dev., 5:1538-1552). The CD experiments presented hereinindicated that 3heptad-F heterodimerized with C/EBP with K_(d)=4×10⁻¹¹M. Therefore, an equimolar mixture of DNA, C/EBP dimers and 3heptad-Fdimers was expected to prevent C/EBP from binding DNA, because of theformation of the C/EBP:3heptad-F heterodimer. The gel shift dataconfirmed that this equimolar mixture completely inhibited C/EBP bindingto a consensus radiolabeled oligonucleotide (FIG. 13A).

[0162] B. Fluorescence

[0163] The second competition assay monitored the intrinsic fluorescenceof the only tryptophan found in the basic region of VBP. DNA binding ofthe VBP-C/EBP chimera increased the fluorescence of this tryptophan byapproximately 40%. The increase in fluorescence is to be expected if thetryptophan is in a more constrained environment, a situation which isexpected upon DNA binding (Cantor and Schimmel, 1980, BiophysicalChemistry. W. H. Freeman and Co., New York), (FIG. 13B). This change inthe intrinsic fluorescence of VBP-C/EBP upon DNA binding allowed theexamination of the kinetic and equilibrium consequences of adding the Fzipper-acidic extension series to this bZIP DNA complex. It was foundthat VBP-C/EBP bound to DNA with a fast on-rate (˜5 seconds), and thatthe addition of the F zipper-acidic extension proteins resulted in adecrease in tryptophan fluorescence. This is expected if the Fzipper-acidic extension proteins heterodimerized with VBP-C/EBP, thuspreventing DNA binding.

[0164] Proteins containing different length acidic extensions appendedto the F zipper were mixed with the VBP-C/EBP DNA complex in a 1 to 1.1molar ratio. The longer acidic extensions caused a greater decrease inthe fluorescence, thus indicating more effective heterodimerization withVBP-C/EBP. Ten additional molar equivalents of DN were added after thereaction came to equilibrium in about 20 minutes. 0heptad-F and1heptad-F were not able to totally displace VBP-C/EBP from DNA, even in11-fold excess. However, at an 11-fold molar excess, 2heptad-F and3heptad-F were able to totally displace VBP-C/EBP from DNA. 4heptad-Fwas the most potent, as 1.1 molar equivalents displaced 85% of VBP-C/EBPfrom its binding to DNA.

[0165] The location of the tryptophan residue in the VBP basic region isfour heptads from the leucine zipper. The tryptophan would be expectedto be in a random coil conformation as a heterodimer with the shorteracidic extensions, such as those having one, two, or three heptadextensions (i.e., 0-3heptad-F), but would be expected to be in a coiledcoil conformation as a heterodimer with more acidic extensions (i.e.,4heptad-F). The tryptophan would be expected to be part of thehydrophobic interface, i.e., it would occupy the a position, and,consequently, might have a greater fluorescence. This was observed. Thetryptophan fluorescence of VBP-C/EBP in mixtures with 0, 1, 2, and3heptad-F is similar to that of VBP-C/EBP alone which we interpret asthe tryptophan fluorescence in the random coil state. In the mixturewith 4-heptad-F, the fluorescence of VBP-C/EBP increases by about 10%suggesting that the tryptophan is more constrained as would happen if itis in the hydrophobic interface of a coiled coil

[0166] C. Transient Transfections

[0167] The third competition assay involved the transient transfectionof constructs harboring the acidic extension and bZIP DNA sequences intoa hepatoma cell line (HepG2) to monitor the transactivation propertiesof C/EBP on a promoter containing a single C/EBP binding site (see alsoExample 1F). C/EBP was shown to be able to transactivate this promoter10-fold (FIG. 14). Constructs containing four, different, potential DNswere also able to inhibit C/EBP transactivation in a 15 to 1 ratio.These four DN's are a truncation of the transactivation domain (ΔC/EBP),0heptad-F, 3heptad-F, and 3heptad-C/EBP. Constructs carrying ΔC/EBP,0heptad-F, and 3heptad-C/EBP inhibited C/EBP transactivation onlyslightly. In marked contrast, 3heptad-F was able to inhibit completelyC/EBP-mediated transactivation. This total inhibition was also observedat a ratio of C/EBP plasmid to 3heptad-F plasmid of 1 to 1. Thus, boththe heterodimerizing F zipper and the acidic extension were discoveredto be essential for total inactivation of C/EBP. In addition, the3heptad-F-containing construct was found to inhibit the transactivationactivity of all the C/EBP family members examined (C/EBPα, C/EBPβ,C/EBPδ).

[0168] To investigate if 3heptad-F inhibition of C/EBP transactivationwas dependent on the C/EBP leucine zipper, C/EBP transactivatingproteins were generated in which the C/EBP leucine zipper was replacedwith the GCN4 leucine zipper. This chimeric protein was able totransactivate a minimal promoter in a C/EBP cis element-dependentmanner. This transactivation was not inhibited by 3heptad-F, thusindicating that the DN properties of 3heptad-F are dependent on specificleucine zipper interactions.

Example 9

[0169] The Acidic Extension Forms a Coiled Coil with the Basic Region ofLeucine Zipper Containing Proteins, e.g., bZIP

[0170] It was found that the addition of one or more heptads of acidicamino acid sequences onto the N-terminus of the leucine zipperstabilized heterodimer formation, as determined by circular dichroism(CD) spectra and thermal melting assays as described herein. This acidicprotein (or polypeptide or peptide) extension formed a heterodimericcoiled coil with the basic region of the wild type or native proteincontaining the leucine zipper and allowed the creation of a dominantnegative to the native protein that stoichiometrically displaced thenative protein from DNA and inhibited transactivation by the nativeprotein.

[0171] Sedimentation equilibrium analysis was carried out in order todetermine the oligomerization state of an equimolar mixture of C/EBP and3heptad-F (FIG. 11). The data were fitted to a single molecular species.The calculated molecular weight for C/EBP is 33,000 Daltons (dimer MW isabout 32,208) and for the C/EBP−3heptad-F mixture is 28,000 (heterodimerMW is about 26,200). These data are consistent with C/EBP forming aheterodimer when mixed with 3heptad-F.

[0172] Circular dichroism (CD) was used to determine the amount ofα-present in mixtures of C/EBP and different length acidic extensionsappended to the F zipper (FIG. 12A) The mixtures of C/EBP withprogressively longer acidic extensions showed a progressive increase inα-content. The increase in α-helical content of the heterodimerizingmixtures as described herein reveals that the leucine zipperdimerization interface is extending into the basic region: the longerthe acidic extension, the farther the coiled coil structure extends fromthe leucine zipper into the basic region. The CD spectra of the acidicextensions series (0heptad-F to 3-heptad-F) as homodimers at 6° C.displayed a similar amount of α-helical content, thus indicating thatthe progressively longer acidic extensions were not helical. The CD meltof 4heptad-F was biphasic; this is believed to indicate that the acidicextension forms a coiled coil which melts at 13° C., independently ofthe leucine zipper.

[0173] The stability of the mixtures of C/EBP and the different lengthacidic extensions was determined by CD thermal melts (Table 2 and FIG.12B). All mixtures had a higher T_(m) than the individual homodimers,thereby suggesting that heterodimers were formed. The 0heptad-F:C/EBPheterodimer was more stable than the C/EBP homodimer, but the former didnot contain any additional helical content. This suggests more stabilityfor the leucine zipper structure. The progressively longer acidicextensions created progressively more stable complexes. These extensionsraised the T_(m) about 10° C., which corresponded to 2.5 kcal/mol orover 100 fold stabilization.

[0174] The energetic and structural consequences of adding up to fourheptads of acidic protein sequence onto the N-terminus of the F leucinezipper was assayed by circular dichroism (CD) spectra and thermal melts.The first acidic heptad appended to the F zipper stabilized theheterodimer with C/EBP by over 1 kcal/mol. This increase in stabilitywas accompanied by an increase in ellipticity, indicative ofα-formation. Each additional acidic heptad extension produced anincrease in ellipticity consistent with extending the leucine zippercoiled coil by one heptad. However, the contribution to stability becamesuccessively less with increased heptad extensions. The combination ofthe increase in ellipticity and the progressive decrease in thecontributed stability suggests that the coiled coil structure betweenthe C/EBP basic region and the acidic extensions is weaker as thestructure extends further from the leucine zipper. Similar results wereobtained if the C/EBP basic region was replaced with other basicregions, e.g., the VBP or GBF basic regions. On the basis of thesefindings, extensions can be designed and created to dimerizespecifically with a subset of basic regions in bZIP proteins andstructurally similar proteins.

Example 10

[0175] Stability of the Heterodimerizing Zipper (F) with C/EBP

[0176] The thermal stability of the heterodimers formed between C/EBPand the F leucine zipper produced as described by C. Vinson et al.,1993, Ibid., either as a chimera with the C/EBP basic region (C/EBP-F)or alone (0heptad-F), was determined (see Table 1 and FIGS. 9C-9D).Using gel shift conditions (50 mM KCl), a chimera containing the Fzipper and the C/EBP basic region (C/EBP-F) formed a heterodimer withC/EBP that was five times more stable than the C/EBP homodimer. However,using more physiological salt conditions (150 mM KCl), C/EBP-F formed aheterodimer with C/EBP that was only twice as stable as the C/EBPhomodimer. Deletion of the basic region (0heptad-F) increased thestability of the heterodimer with C/EBP by 0.5 kcal/mol, but theheterodimer was still not as stable as the wild type C/EBP homodimerbound to DNA. These results showed that initial attempts to create a DNthat could displace C/EBP from DNA by making modifications only in theleucine zipper were unsuccessful.

[0177] The wild type C/EBP homodimer was less stable by 4° C. in 50 mMKCl compared with 150 mM KCl, presumably because of repulsion betweenthe basic regions. This decrease was not observed for a C/EBP:C/EBP-Fheterodimer, because the increase in the electrostatic attractionbetween the F zipper and the wild type C/EBP zipper apparentlycompensated for the increased repulsion between the two C/EBP basicregions. Similarly, the increased stability of the C/EBP:0heptad-Fheterodimer relative to that of the C/EBP:C/EBP-F heterodimer may havebeen due to the same interactions, because the 0heptad-F mutant lackedthe repulsive basic region.

Example 11

[0178] The Acidic Extension Forms a Coiled Coil with Two Additional bZIPBasic Regions

[0179] To investigate if the acidic N-terminal extension formed a coiledcoil with other bZIP basic regions, chimeras were generated containingtwo additional basic regions attached to the native C/EBP leucinezipper. The first basic region was derived from the chicken vitellogeningene-binding protein (VBP) (S. Iyer et al., 1991, Mol. Cell. Biol.,11:4863-4875), the chicken equivalent of TEF (D. Drolet et al., 1991,Genes Dev., 5:1739-1753), and the second was derived from GBF-1, a plantbZIP protein known to bind to the most divergent cis element of the bZIPproteins (U. Schindler et al., 1992, EMBO J., 11:1261-1273). The resultsof thermal melts of these chimeras (i.e., VBP-C/EBP and GBF-C/EBP) ashomodimers showed a T_(m) within 1° C. of C/EBP, indicating thatdimerization strength was determined by the leucine zipper (Table 2).Table 2 presents the stability of heterodimers between the F zipper withdifferent length acidic extensions and chimeras containing threedifferent bZIP basic regions appended to the C/EBP zipper.

[0180] As shown, four acidic heptad extensions were able to interactfavorably with the three different bZIP basic regions that wereexamined. Initially, this was found to be surprising since it wasassumed that the C/EBP basic region would be a better candidate forforming a coiled coil with the acidic extension than the GBF and VBPbasic regions. Most bZIP proteins, including GBF and VBP, contain abasic amino acid in the first d position N-terminal of the leucinezipper, while C/EBP contains an alanine. It was expected that the basicamino acids would disfavor coiled coil formation while the alanine wouldbe more favorable. CD thermal melts indicated that this was true; C/EBPheterodimerized with 1heptad-F 1.7±0.5 kcal/mol better than with0heptad-F, while the VBP and GBF basic regions were only stabilized0.9±0.5 and 1.3±0.5 kcal/mol, respectively. Interestingly, thisdifference in stability among the three basic region became lesspronounced as the basic region was “zippered” up by longer acidicextensions.

[0181] The acidic extension to the leucine zipper domain wassuccessfully stabilized by a variety of different basic regions, thussuggesting that this protein sequence can be used to create dominantnegatives for a variety of different types of proteins comprised ofbasic domains and leucine zipper domains. The biological assaysexemplified herein demonstrate that C/EBP transcription factor proteinsdesigned and produced to contain the acidic extension are robustdominant negatives of the C/EBP family, which is considered to berepresentative of other protein members in the bZIP family or instructurally similar protein families. TABLE 2 Dominant bZIP proteinnegative Homodimer C/EBP-C/EBP VBP-C/EBP GBF-C/EBP (DN) T_(m)(ΔG)k_(d)T_(m)(ΔG)k_(d) T_(m)(ΔG)k_(d) T_(m)(ΔG)k_(d) Homodimer 49.5 (−10.9)1e−850.0(−11.1)8e−9 50.2(−10.6)2e−8 Heterodimer with 0heptad-F30.0(−8.0)1e−6 55.4(−11.8)3e−9 55.1(−12.2)1e−9 55.7(−11.9)2e−9 1heptad-F29.0(−8.0)1e−6 60.8(−13.5)1e−10 60.5(−13.1)3e−10 62.0(−13.2)2e−102heptad-F 24.8(−7.4)4e−6 63.6(−13.9)7e−11 62.8(−13.8)9e−1163.1(−13.6)1e−10 3heptad-F 28.3(−7.9)2e−6 63.8(−14.3)4e−1164.2(−14.7)2e−11 64.0(−14.2)4e−11 4heptad-F 15.2(−6.3)2e−564.8(−14.2)4e−11 65.2(−14.8)1e−11 65.0(−14.5)3e−11 3heptad-C/EBP61.0(−12.6)6e−10

[0182] The two chimeras were mixed with the four different length acidicextensions appended to the F zipper and their stability was determinedfrom CD thermal melts. Interestingly, the two additional basic regionswere also stabilized by the acidic extensions. The degree ofstabilization was the same for the three basic regions examined, thussuggesting that the interaction occurred with the more conserved part ofthe basic region.

[0183] Thus, two lines of evidence suggested that the acidic extensionformed a coiled coil with the basic region in the proteins expressed andassayed in accordance with the invention. The first was that inα-helical content increased when the acid extensions were mixed with thebasic regions to form chimeric structures. The second was determinedfrom the fluorescence analysis of exemplary heterodimers formed betweenchimeras of the VBP and C/EBP bZIP proteins and the F zipper-acidicextension series (See Examples 1E and 8B). The tryptophan in theVBP-C/EBP chimera is located in the basic region, four heptads from theleucine zipper. If a coiled coil were formed between the basic region ofVBP and the acidic extension, then only the heterodimer between the4heptad-F and VBP-C/EBP would be expected to place the tryptophan in thecoiled coil structure, thereby causing a new fluorescence signal.Indeed, this result was observed. However, such a change in thefluorescence signal was not observed in heterodimers which contained theshorter acidic extensions.

Example 12

[0184] The Acidic Extension is a DNA Mimetic

[0185] The importance of different coiled coil positions (a, b, c, d, e,f, g) in the acidic extension to heterodimer stability with native C/EBPwas examined by producing four mutant proteins. Each protein comprised asubset of the mutations to the C/EBP basic region that was used tocreate 3heptad-F. The largest contribution to heterodimer stability wasprovided by the glutamic acids placed in the e and g positions. Thesenegatively charged amino acids presumably interact with the positivelycharged amino acids found in abundance in the basic region, thussuggesting that the acidic extension acts as a DNA mimetic by matchingthe electrostatic properties of DNA. The three additional mutantproteins demonstrated the contributions of the N-terminal helical capconsisting of three glycines, the cap plus the hydrophobic core (a andd), and the cap plus general electrostatic and forming concerns (b, andc). All of the mutant proteins contributed some stability to theheterodimer with the wild type C/EBP basic region (see Example 1F).

Example 13

[0186] Transgenic Mouse Studies

[0187] Transgenic animals, and in particular mice, are created using theconstructs described for the transient transfection assays in Examples1F and 8C. Briefly, the constructs are introduced into an animal or anancestor of the animal at an embryonic stage, i.e., the one-cell stage,or generally not later than about the eight-cell stage. Transgenicanimals carrying the constructs of the invention can be made by severalmethods known to those having skill in the art. One method involvestransfecting a retrovirus constructed to contain the DNA sequenceencoding a dominant negative leucine-zipper containing protein toprovide a complete shuttle vector harboring the dominant negativenucleic acid sequence as a transgene. Another method involves directlyinjecting a transgene into the embryo. A third method involves the useof embryonic stem cells. Examples of animals into which the transgenesor constructs may be introduced include, but are not limited to, mice,rats, other rodents, sheep, pigs, and primates (see “The Introduction ofForeign Genes into Mice” and the cited references therein, In:Recombinant DNA, Eds. J. D. Watson et al., W. H. Freeman and Company,New York, New York, pp. 254-272). Transgenic animals carrying and stablyexpressing transgenes encoding the DNs of the invention can be used asbiological models for the study of cancer and tumor regression oratrophy, for example. The appropriate tissues of the transgenic animalcan be monitored for the integration of the construct, or componentsthereof, by assaying for the presence of RNA or DNA in the cells usingestablished methods in the art.

[0188] In addition, transgenic mice can be produced in which tissuespecific promoters are used in constructs containing DNA sequencesencoding acidic extensions to the zipper of bZIP, or structurallysimilar proteins, as well as other pertinent sequences, to act asdominant negatives to one or more of the three C/EBP isoforms expressedduring adipose conversion of 3T3-L1 cells (Z. Cao et al., Genes Dev.,5:1538-1552). The production of functional dominant negatives in thissystem will allow the inactivation of gene(s), or mutants thereof,involved in the conversion of cells to adipose cells. The constructs asdescribed may provide the potential to reduce or alleviate theconditions of morbid obesity and type II diabetes associated with thesyndrome of morbid obesity in model animals and in humans.

[0189] Tissue specific promoters such as the whey activating protein(WAP) which targets mammary tissue, can be used to create transgenicanimals in which the DN is localized to and functions specifically inbreast tissues. For example, a construct can be designed to include themouse WAP promoter and isolated DNA encoding the DN proteins of theinvention, in addition to the appropriate elements for propertranscription of the gene and protein expression in cells. The activityof the DN to inactivate the abnormal activity of cellular proteins canbe monitored in mammary tissue of the female transgenic mice.

[0190] In addition, the constructs can be used in human gene therapytreatments as known in the art to inactivate cellular protein productsthe regulation of which is controlled by or linked to transcriptionregulatory proteins possessing leucine zipper structures. Gene therapytechniques which can be modified as required for use of the constructsof the invention are described, for example, in U.S. Pat. No. 5,399,346to W. French Anderson et al.

Example 14

[0191] Transgenic Mouse Studies Using a Dominant Negative C/EBP toCreate Phenotypically “Skinny” Mice

[0192] As set forth in Example 13, transgenic mouse studies were carriedout and mouse pups were born which exhibited a transgenic phenotypeshowing that the dominant negatives produced and expressed in accordancewith the invention functioned in an in vivo environment in an animal.The transgenic mice were produced by routine methods practiced in theart of transgenic animal production.

[0193] In these studies, transgenic mice were produced by injectingearly-stage mouse embryos with the construct described in FIG. 30. Asdescribed, this plasmid construct was designed to contain the 422/aP2promoter (adipose fatty acid-binding protein), DNA sequence encoding thedominant negative 3heptadF C/EBP of the invention, which produces, upontranslation and expression, an acidically extended bZIP C/EBP protein,that functions as a dominant negative to the non-mutant C/EBP protein,and other regulatory sequences as described for FIG. 30. C/EBP has beenimplicated in the development and differentiation of adipose (fat)cells. The dominant negative 3heptadF C/EBP sequence was placed underthe control of the 422/aP2 adipocyte-specific promoter, and alsocontained the FLAGφ10 epitope as described hereinabove.

[0194] Four initial founder mice were produced by injecting mouseembryos with the construct capable of expressing the dominant negativeC/EBP protein. The founder mice were used to produce offspring carryingone or more copies of the transgene. One of the offspring of a foundermouse showed a thin, skinny phenotype, which would be expected ifexpression of the dominant negative affected the expression of normalC/EBP protein and resulted in negative regulation and influence ofadipose or fat cell and tissue development and differentiation.

[0195] More specifically, the male founder transgenic mouse impregnatedtwo female mice. The two litters produced by these two females containedtwo phenotypically distinct types of pups: the first types were scrawny,small-sized mice with scruffy fur and the second types were normal-sizedwith normal-looking fur. The small-sized, scrawny and scruffy pups allcarried copies of the transgene, while their normal littermates did not.These results indicate that constructs designed to contain DNA encodingacidically extended dominant negatives and expressed as transgenes inanimals in vivo can influence, e.g., via inactivation, the expressionand/or function of a normal counterpart protein encoded by the normalgene. In addition, such dominant negatives can demonstrably affect thephenotypes of the animals carrying the transgene, which were bred fromthe transgenic founder animals.

[0196] The contents of the patents, articles, texts, and referencescontained herein are hereby incorporated by reference in their entirety.

[0197] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope thereof, as described in the specification andas defined in the appended claims.

1 64 24 amino acids amino acid unknown linear peptide 1 Asp Pro Asp LeuGlu Lys Glu Ala Glu Glu Leu Glu 1 5 10 Gln Glu Asn Ala Glu Leu Glu LeuGlu Asp Ser Phe 15 20 25 amino acids amino acid unknown linear peptide 2Asp Pro Asp Leu Glu Lys Glu Ala Glu Glu Leu Glu 1 5 10 Gln Glu Asn AlaGlu Leu Glu Glu Leu Glu Asp Ser 15 20 Phe 25 26 amino acids amino acidunknown linear peptide 3 Asp Pro Asp Leu Glu Lys Glu Ala Glu Glu Leu Glu1 5 10 Gln Glu Asn Ala Glu Leu Glu Glu Glu Leu Glu Asp 15 20 Ser Phe 2519 amino acids amino acid unknown linear peptide 4 Asp Pro Asp Glu GluGlu Asp Asp Glu Glu Glu Leu 1 5 10 Glu Glu Leu Glu Asp Ser Phe 15 11amino acids amino acid unknown linear peptide 5 Asp Pro Asp Leu Glu GluLeu Glu Asp Ser Phe 1 5 10 223 base pairs nucleic acid unknown linearDNA (genomic) 6 GGATCCCCTT CCTACACAGC CTGCTGAAGA AGCAGCACGA 40AAGAGAGAGG TTCGTCTAAT GAAGAACAGG GAAGCAGCAA 80 GAGAATGTCG TAGAAAGAAGAAAGAATATG TGAAATGTTT 120 AGAGAACAGA GTGGCAGTGC TTGAAAACCA AAACAAAACA160 TTGATTGAGG AGCTAAAAGC ACTTAAGGAC CTTTACTGCC 200 ACAAGTCAGATTAATTCAAG CTT 223 68 amino acids amino acid unknown linear peptide 7Leu Pro Thr Gln Pro Ala Glu Glu Ala Ala Arg Lys 1 5 10 Arg Glu Val ArgLeu Met Lys Asn Arg Glu Ala Ala 15 20 Arg Glu Cys Arg Arg Lys Lys LysGlu Tyr Val Lys 25 30 35 Cys Leu Glu Asn Arg Val Ala Val Leu Glu Asn Gln40 45 Asn Lys Thr Leu Ile Glu Glu Leu Lys Ala Leu Lys 50 55 60 Asp LeuTyr Cys His Lys Ser Asp 65 290 base pairs nucleic acid unknown linearDNA (genomic) 8 CCATGGACTA CAAGGACGAC GATGACAAGC ATATGGCTAG 40CATGACTGGT GGACAGCAAA TGGGTCGGGA TCCCCTTCCT 80 ACACAGCCTG CTGAAGAAGCAGCACGAAAG AGAGAGGTTC 120 GTCTAATGAA GAACAGGGAA GCAGCAAGAG AATGTCGTAG160 AAAGAAGAAA GAATATGTGA AATGTTTAGA GAACAGAGTG 200 GCAGTGCTTGAAAACCAAAA CAAAACATTG ATTGAGGAGC 240 TAAAAGCACT TAAGGACCTT TACTGCCACAAGTCAGATTA 280 ATTCAAGCTT 290 92 amino acids amino acid unknown linearpeptide 9 Met Asp Tyr Lys Asp Asp Asp Asp Lys His Met Ala 1 5 10 Ser MetThr Gly Gly Gln Gln Met Gly Arg Asp Pro 15 20 Leu Pro Thr Gln Pro AlaGlu Glu Ala Ala Arg Lys 25 30 35 Arg Glu Val Arg Leu Met Lys Asn Arg GluAla Ala 40 45 Arg Glu Cys Arg Arg Lys Lys Lys Glu Tyr Val Lys 50 55 60Cys Leu Glu Asn Arg Val Ala Val Leu Glu Asn Gln 65 70 Asn Lys Thr LeuIle Glu Glu Leu Lys Ala Leu Lys 75 80 Asp Leu Tyr Cys His Lys Ser Asp 8590 266 base pairs nucleic acid unknown linear DNA (genomic) 10CCATGGACTA CAAGGACGAC GATGACAAGC ATATGGCTAG 40 CATGACTGGT GGACAGCAAATGGGTCGGGA TCCTGACCTG 80 GAACAACGTG CTGAGGAACT GGCCCGTGAA AACGAAGAGC 120TGGAAAAAGA GGCCGAAGAG CTGGAGCAGG AACTGGCAGA 160 ACTCGAGAAC AGAGTGGCAGTGCTTGAAAA CCAAAACAAA 200 ACATTGATTG AGGAGCTAAA AGCACTTAAG GACCTTTACT240 GCCACAAGTC AGATTAATTC AAGCTT 266 84 amino acids amino acid unknownlinear peptide 11 Met Asp Tyr Lys Asp Asp Asp Asp Lys His Met Ala 1 5 10Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Pro 15 20 Asp Leu Glu GlnArg Ala Glu Glu Leu Ala Arg Glu 25 30 35 Asn Glu Glu Leu Glu Lys Glu AlaGlu Glu Leu Glu 40 45 Gln Glu Leu Ala Glu Leu Glu Asn Arg Val Ala Val 5055 60 Leu Glu Asn Gln Asn Lys Thr Leu Ile Glu Glu Leu 65 70 Lys Ala LeuLys Asp Leu Tyr Cys His Lys Ser Asp 75 80 262 base pairs nucleic acidunknown linear DNA (genomic) 12 GGATCCCAAG GTGGAACAGT TATCTCCAGAAGAAGAAGAG 40 AAAAGGAGAA TCCGAAGGGA AAGGAATAAG ATGGCTGCAG 80 CCAAATGCCGCAACCGGAGG AGGGAGCTGA CTGATACACT 120 CCAAGCGGAG ACAGACCAAC TAGAAGATGAGAAGTCTGCT 160 TTGCAGACCG AGATTGCCAA CCTGCTGAAG GAGAAGGAAA 200AACTAGAGTT CATCCTGGCA GCTCACCGAC CTGCCTGCAA 240 GATCCCTGAT TAATTCAAGC TT262 82 amino acids amino acid unknown linear peptide 13 Pro Asp Lys ValGlu Gln Leu Ser Pro Glu Glu Glu 1 5 10 Glu Lys Arg Arg Ile Arg Arg GluArg Asn Lys Met 15 20 Ala Ala Ala Lys Cys Arg Asn Arg Arg Arg Glu Leu 2530 35 Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Leu Glu 40 45 Asp Glu LysSer Ala Leu Gln Thr Glu Ile Ala Asn 50 55 60 Leu Leu Lys Glu Lys Glu LysLeu Glu Phe Ile Leu 65 70 Ala Ala His Arg Pro Ala Cys Lys Ile Pro 75 80324 base pairs nucleic acid unknown linear DNA (genomic) 14 CCATGGACTACAAGGACGAC GATGACAAGC ATATGGCTAG 40 CATGACTGGT GGACAGCAAA TGGGTCGGGATCCCAAGGTG 80 GAACAGTTAT CTCCAGAAGA AGAAGAGAAA AGGAGAATCC 120 GAAGGGAAAGGAATAAGATG GCTGCAGCCA AATGCCGCAA 160 CCGGAGGAGG GAGCTGACTG ATACACTCCAAGCGGAGACA 200 GACCAACTAG AAGATGAGAA GTCTGCTTTG CAGACCGAGA 240TTGCCAACCT GCTGAAGGAG AAGGAAAAAC TAGAGTTCAT 280 CCTGGCAGCT CACCGACCTGCCTGCAAGAT CCCTGATTAA 320 GCTT 324 105 amino acids amino acid unknownlinear peptide 15 Met Asp Tyr Lys Asp Asp Asp Asp Lys His Met Ala 1 5 10Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Pro 15 20 Lys Val Glu GlnLeu Ser Pro Glu Glu Glu Glu Lys 25 30 35 Arg Arg Ile Arg Arg Glu Arg AsnLys Met Ala Ala 40 45 Ala Lys Cys Arg Asn Arg Arg Arg Glu Leu Thr Asp 5055 60 Thr Leu Gln Ala Glu Thr Asp Gln Leu Glu Asp Glu 65 70 Lys Ser AlaLeu Gln Thr Glu Ile Ala Asn Leu Leu 75 80 Lys Glu Lys Glu Lys Leu GluPhe Ile Leu Ala Ala 85 90 95 His Arg Pro Ala Cys Lys Ile Pro Asp 100 105281 base pairs nucleic acid unknown linear DNA (genomic) 16 ATATACATATGGCTAGCATG ACTGGTGGAC AGCAAATGGG 40 TCGGGATCCT GACCTGGAAC AACGTGCTGAGGAACTGGCC 80 CGTGAAAACG AAGAGCTGGA AAAAGAGGCC GAAGAGCTGG 120 AGCAGGAAAACGCTGAACTC GAGGCGGAGA CAGACCAACT 160 AGAAGATGAG AAGTCTGCTT TGCAGACCGAGATTGCCAAC 200 CTGCTGAAGG AGAAGGAAAA ACTAGAGTTC ATCCTGGCAG 240CTCACCGACC TGCCTGCAAG ATCCCTGATT AATTCAAGCT 280 T 281 86 amino acidsamino acid unknown linear peptide 17 Met Ala Ser Met Thr Gly Gly Gln GlnMet Gly Arg 1 5 10 Asp Pro Asp Leu Glu Gln Arg Ala Glu Glu Leu Ala 15 20Arg Glu Asn Glu Glu Leu Glu Lys Glu Ala Glu Glu 25 30 35 Leu Glu Gln GluAsn Ala Glu Leu Glu Ala Glu Thr 40 45 Asp Gln Leu Glu Asp Glu Lys SerAla Leu Gln Thr 50 55 60 Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu65 70 Glu Phe Ile Leu Ala Ala His Arg Pro Ala Cys Lys 75 80 Ile Pro 85300 base pairs nucleic acid unknown linear DNA (genomic) 18 CCATGGACTACAAGGACGAC GATGACAAGC ATATGGCTAG 40 CATGACTGGT GGACAGCAAA TGGGTCGGGATCCTGACCTG 80 GAACAACGTG CTGAGGAACT GGCCCGTGAA AACGAAGAGC 120 TGGAAAAAGAGGCCGAAGAG CTGGAGCAGG AAAACGCTGA 160 ACTCGAGGCG GAGACAGACC AACTAGAAGATGAGAAGTCT 200 GCTTTGCAGA CCGAGATTGC CAACCTGCTG AAGGAGAAGG 240AAAAACTAGA GTTCATCCTG GCAGCTCACC GACCTGCCTG 280 CAAGATCCCT GATTAAGCTT300 97 amino acids amino acid unknown linear peptide 19 Met Asp Tyr LysAsp Asp Asp Asp Lys His Met Ala 1 5 10 Ser Met Thr Gly Gly Gln Gln MetGly Arg Asp Pro 15 20 Asp Leu Glu Gln Arg Ala Glu Glu Leu Ala Arg Glu 2530 35 Asn Glu Glu Leu Glu Lys Glu Ala Glu Glu Leu Glu 40 45 Gln Glu AsnAla Glu Leu Glu Ala Glu Thr Asp Gln 50 55 60 Leu Glu Asp Glu Lys Ser AlaLeu Gln Thr Glu Ile 65 70 Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu GluPhe 75 80 Ile Leu Ala Ala His Arg Pro Ala Cys Lys Ile Pro 85 90 95 Asp353 base pairs nucleic acid unknown linear DNA (genomic) 20 CCATGGACTACAAGGACGAC GATGACAAGC ATATGGCTAG 40 CATGACTGGT GGACAGCAAA TGGGTCGGGATCCCTCCCCT 80 ATTGACATGG AGTCGCAGGA GAGAATCAAA GCCGAGAGAA 120 AACGCATGAGAAACAGAATT GCGGCGTCCA AATGCCGGAA 160 AAGGAAGTTG GAAAGGATTG CCAGGTTGGAAGAAAAAGTG 200 AAAACTTTGA AAGCCCAGAA CTCAGAGCTG GCATCCACGG 240CCAACATGCT CAGAGAACAG GTTGCACAGC TTAAGCAGAA 280 GGTCATGAAC CATGTCAACAGCGGGTGCCA GCTAATGCTA 320 ACACAACAGT TGCAAACGTT TTGATTCAAG CTT 353 113amino acids amino acid unknown linear peptide 21 Met Asp Tyr Lys Asp AspAsp Asp Lys His Met Ala 1 5 10 Ser Met Thr Gly Gly Gln Gln Met Gly ArgAsp Pro 15 20 Ser Pro Ile Asp Met Glu Ser Gln Glu Arg Ile Lys 25 30 35Ala Glu Arg Lys Arg Met Arg Asn Arg Ile Ala Ala 40 45 Ser Lys Cys ArgLys Arg Lys Leu Glu Arg Ile Ala 50 55 60 Arg Leu Glu Glu Lys Val Lys ThrLeu Lys Ala Gln 65 70 Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg 7580 Glu Gln Val Ala Gln Leu Lys Gln Lys Val Met Asn 85 90 95 His Val AsnSer Gly Cys Gln Leu Met Leu Thr Gln 100 105 Gln Leu Gln Thr Phe 110 324base pairs nucleic acid unknown linear DNA (genomic) 22 ATACATATGGCTAGCATGAC TGGTGGACAG CAAATGGGTC 40 GGGATCCCGA CGAAGAGGAA GATGACGAAGAAGAACTCGA 80 GGAACTGGAA GACAGCTTTC ACAGTTTGCG GGACTCAGTC 120 CCATCACTCCAAGGAGAGAA GGCATCCCGG GCCCAAATCC 160 TAGACAAAGC AACAGAGTAT ATCCAGTATATGCGAAGGAA 200 AAACCATACG CACCAGCAAG ACATTGATGA CCTCAAGCGG 240CAGAATGCTC TTCTGGAGCA ACAAGTCCGT GCACTGGAGA 280 AGGCAAGATC AAGTGCCCAACTGCAGACCT GAGGCAAGCT 320 TATC 324 101 amino acids amino acid unknownlinear peptide 23 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 1 5 10Asp Pro Asp Glu Glu Glu Asp Asp Glu Glu Glu Leu 15 20 Glu Glu Leu GluAsp Ser Phe His Ser Leu Arg Asp 25 30 35 Ser Val Pro Ser Leu Gln Gly GluLys Ala Ser Arg 40 45 Ala Gln Ile Leu Asp Lys Ala Thr Glu Tyr Ile Gln 5055 60 Tyr Met Arg Arg Lys Asn His Thr His Gln Gln Asp 65 70 Ile Asp AspLeu Lys Arg Gln Asn Ala Leu Leu Glu 75 80 Gln Gln Val Arg Ala Leu GluLys Ala Arg Ser Ser 85 90 95 Ala Gln Leu Gln Thr 100 341 base pairsnucleic acid unknown linear DNA (genomic) 24 ATATACATAT GGCTAGCATGACTGGTGGAC AGCAAATGGG 40 TCGGGATCCT GACCTGGAAA AAGAGGCCGA AGAGCTGGAG 80CAGGAAAACG CTGAACTCGA GCTGGAAGAC AGCTTTCACA 120 GTTTGCGGGA CTCAGTCCCATCACTCCAAG GAGAGAAGGC 160 ATCCCGGGCC CAAATCCTAG ACAAAGCAAC AGAGTATATC200 CAGTATATGC GAAGGAAAAA CCATACGCAC CAGCAAGACA 240 TTGATGACCTCAAGCGGCAG AATGCTCTTC TGGAGCAACA 280 AGTCCGTGCA CTGGAGAAGG CAAGATCAAGTGCCCAACTG 320 CAGACCTGAG GCAAGCTTAT C 341 106 amino acids amino acidunknown linear peptide 25 Met Ala Ser Met Thr Gly Gly Gln Gln Met GlyArg 1 5 10 Asp Pro Asp Leu Glu Lys Glu Ala Glu Glu Leu Glu 15 20 Gln GluAsn Ala Glu Leu Glu Leu Glu Asp Ser Phe 25 30 35 His Ser Leu Arg Asp SerVal Pro Ser Leu Gln Gly 40 45 Glu Lys Ala Ser Arg Ala Gln Ile Leu AspLys Ala 50 55 60 Thr Glu Tyr Ile Gln Tyr Met Arg Arg Lys Asn His 65 70Thr His Gln Gln Asp Ile Asp Asp Leu Lys Arg Gln 75 80 Asn Ala Leu LeuGlu Gln Gln Val Arg Ala Leu Glu 85 90 95 Lys Ala Arg Ser Ser Ala Gln LeuGln Thr 100 105 344 base pairs nucleic acid unknown linear DNA (genomic)26 ATATACATAT GGCTAGCATG ACTGGTGGAC AGCAAATGGG 40 TCGGGATCCT GACCTGGAAAAAGAGGCCGA AGAGCTGGAG 80 CAGGAAAACG CTGAACTCGA GGAACTGGAA GACAGCTTTC 120ACAGTTTGCG GGACTCAGTC CCATCACTCC AAGGAGAGAA 160 GGCATCCCGG GCCCAAATCCTAGACAAAGC AACAGAGTAT 200 ATCCAGTATA TGCGAAGGAA AAACCATACG CACCAGCAAG240 ACATTGATGA CCTCAAGCGG CAGAATGCTC TTCTGGAGCA 280 ACAAGTCCGTGCACTGGAGA AGGCAAGATC AAGTGCCCAA 320 CTGCAGACCT GAGGCAAGCT TATC 344 107amino acids amino acid unknown linear peptide 27 Met Ala Ser Met Thr GlyGly Gln Gln Met Gly Arg 1 5 10 Asp Pro Asp Leu Glu Lys Glu Ala Glu GluLeu Glu 15 20 Gln Glu Asn Ala Glu Leu Glu Glu Leu Glu Asp Ser 25 30 35Phe His Ser Leu Arg Asp Ser Val Pro Ser Leu Gln 40 45 Gly Glu Lys AlaSer Arg Ala Gln Ile Leu Asp Lys 50 55 60 Ala Thr Glu Tyr Ile Gln Tyr MetArg Arg Lys Asn 65 70 His Thr His Gln Gln Asp Ile Asp Asp Leu Lys Arg 7580 Gln Asn Ala Leu Leu Glu Gln Gln Val Arg Ala Leu 85 90 95 Glu Lys AlaArg Ser Ser Ala Gln Leu Gln Thr 100 105 347 base pairs nucleic acidunknown linear DNA (genomic) 28 ATATACATAT GGCTAGCATG ACTGGTGGACAGCAAATGGG 40 TCGGGATCCT GACCTGGAAA AAGAGGCCGA AGAGCTGGAG 80 CAGGAAAACGCTGAACTCGA GGAAGAGCTG GAAGACAGCT 120 TTCACAGTTT GCGGGACTCA GTCCCATCACTCCAAGGAGA 160 GAAGGCATCC CGGGCCCAAA TCCTAGACAA AGCAACAGAG 200TATATCCAGT ATATGCGAAG GAAAAACCAT ACGCACCAGC 240 AAGACATTGA TGACCTCAAGCGGCAGAATG CTCTTCTGGA 280 GCAACAAGTC CGTGCACTGG AGAAGGCAAG ATCAAGTGCC320 CAACTGCAGA CCTGAGGCAA GCTTATC 347 108 amino acids amino acid unknownlinear peptide 29 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 1 5 10Asp Pro Asp Leu Glu Lys Glu Ala Glu Glu Leu Glu 15 20 Gln Glu Asn AlaGlu Leu Glu Glu Glu Leu Glu Asp 25 30 35 Ser Phe His Ser Leu Arg Asp SerVal Pro Ser Leu 40 45 Gln Gly Glu Lys Ala Ser Arg Ala Gln Ile Leu Asp 5055 60 Lys Ala Thr Glu Tyr Ile Gln Tyr Met Arg Arg Lys 65 70 Asn His ThrHis Gln Gln Asp Ile Asp Asp Leu Lys 75 80 Arg Gln Asn Ala Leu Leu GluGln Gln Val Arg Ala 85 90 95 Leu Glu Lys Ala Arg Ser Ser Ala Gln Leu GlnThr 100 105 277 base pairs nucleic acid unknown linear DNA (genomic) 30GGATCCCAAC GACAAGAGGC GGACACACAA CGTCTTGGAA 40 CGTCAGAGGA GGAACGAGCTGAAGCGCAGC TTTTTTGCCC 80 TGCGTGACCA GATCCCTGAA TTGGAAAACA ACGAAAAGGC 120CCCCAAGGTA GTGATCCTCA AAAAAGCCAC CGCCTACATC 160 CTGTCCATTC AAGCAGACGAGCACAAGCTC ACCTCTGAAA 200 AGGACTTATT GAGGAAACGA CGAGAACAGT TGAAACACAA240 ACTCGAACAG CTTCGAAACT CTGGTGCATA AAAGCTT 277 87 amino acids aminoacid unknown linear peptide 31 Asn Asp Lys Arg Arg Thr His Asn Val LeuGlu Arg 1 5 10 Gln Arg Arg Asn Glu Leu Lys Arg Ser Phe Phe Ala 15 20 LeuArg Asp Gln Ile Pro Glu Leu Glu Asn Asn Glu 25 30 35 Lys Ala Pro Lys ValVal Ile Leu Lys Lys Ala Thr 40 45 Ala Tyr Ile Leu Ser Ile Gln Ala AspGlu His Lys 50 55 60 Leu Thr Ser Glu Lys Asp Leu Leu Arg Lys Arg Arg 6570 Glu Gln Leu Lys His Lys Leu Glu Gln Leu Arg Asn 75 80 Ser Gly Ala 85296 base pairs nucleic acid unknown linear DNA (genomic) 32 ATGGCTAGCATGACTGGTGG ACAGCAAATG GGTCGGGATC 40 CTGGCGGTGG CCTGGAACAA CGTGCTGAGGAACTGGCCCG 80 TGAAAACGAA GAGCTGGAAA AAGAGGCCGA AGAGCTGGAG 120 CAGGAAAACGCTGAACTCGA GCAGGAAGTG TTGGAGTTGG 160 AAAGTCGTAA TGACCGCCTG CGCAAGGAAGTGGAACAGCT 200 GGAGCGTGAA CTGGACACGC TGCGGGGTAT CTTCCGCCAG 240CTGCCTGAGA GCTCCTTGGT CAAGGCCATG GGCAACTGCG 280 CGTGAGGCGA ATTCAA 296 13amino acids amino acid unknown linear peptide 33 Ala Ser Met Thr Gly GlyGln Gln Met Gly Arg Asp 1 5 10 Pro 52 amino acids amino acid unknownlinear peptide 34 Gly Gly Gly Thr Gln Gln Glu Val Leu Glu Leu Glu 1 5 10Ser Arg Asn Asp Arg Leu Arg Lys Glu Val Glu Gln 15 20 Leu Glu Arg GluLeu Asp Thr Leu Arg Gly Ile Phe 25 30 35 Arg Gln Leu Pro Glu Ser Ser LeuVal Lys Ala Met 40 45 Gly Asn Cys Ala 50 12 amino acids amino acidunknown linear peptide 35 Gly Gly Gly Leu Glu Gln Glu Asn Ala Glu LeuGlu 1 5 10 19 amino acids amino acid unknown linear peptide 36 Gly GlyGly Leu Glu Lys Glu Ala Glu Glu Leu Glu 1 5 10 Gln Glu Asn Ala Glu LeuGlu 15 26 amino acids amino acid unknown linear peptide 37 Gly Gly GlyLeu Ala Arg Glu Asn Glu Glu Leu Glu 1 5 10 Lys Glu Ala Glu Glu Leu GluGln Glu Asn Ala Glu 15 20 Leu Glu 25 33 amino acids amino acid unknownlinear peptide 38 Gly Gly Gly Leu Glu Gln Arg Ala Glu Glu Leu Ala 1 5 10Arg Glu Asn Glu Glu Leu Glu Lys Glu Ala Glu Glu 15 20 Leu Glu Gln GluAsn Ala Glu Leu Glu 25 30 26 amino acids amino acid unknown linearpeptide 39 Gly Gly Gly Leu Ala Arg Asn Asn Ile Ala Val Arg 1 5 10 LysSer Arg Asp Lys Ala Lys Gln Arg Asn Val Glu 15 20 Leu Glu 25 26 aminoacids amino acid unknown linear peptide 40 Gly Gly Gly Leu Ala Arg AsnAsn Ile Ala Leu Arg 1 5 10 Lys Ser Ala Asp Lys Leu Lys Gln Arg Asn ValGlu 15 20 Leu Glu 25 26 amino acids amino acid unknown linear peptide 41Gly Gly Gly Leu Ala Arg Glu Asn Ile Ala Val Glu 1 5 10 Lys Glu Arg AspLys Ala Glu Gln Glu Asn Val Glu 15 20 Leu Glu 25 26 amino acids aminoacid unknown linear peptide 42 Gly Gly Gly Leu Ala Arg Asn Asn Glu GluVal Arg 1 5 10 Lys Ser Arg Glu Glu Ala Lys Gln Arg Asn Ala Glu 15 20 LeuGlu 25 141 amino acids amino acid unknown linear peptide 43 Ser Glu TyrGln Pro Ser Leu Phe Ala Leu Asn Pro 1 5 10 Met Gly Phe Ser Pro Leu AspGly Ser Lys Ser Thr 15 20 Asn Glu Asn Val Ser Ala Ser Thr Ser Thr AlaLys 25 30 35 Pro Met Val Gly Gln Leu Ile Phe Asp Lys Phe Ile 40 45 LysThr Glu Glu Asp Pro Gly Lys Ala Lys Lys Ser 50 55 60 Val Asp Lys Asn SerAsn Glu Tyr Arg Val Arg Arg 65 70 Glu Arg Asn Asn Ile Ala Val Arg LysSer Arg Asp 75 80 Lys Ala Lys Gln Arg Asn Val Glu Thr Gln Gln Lys 85 9095 Val Leu Glu Leu Thr Ser Asp Asn Asp Arg Leu Arg 100 105 Lys Arg ValGlu Gln Leu Ser Arg Glu Leu Asp Thr 110 115 120 Leu Arg Gly Ile Phe ArgGln Leu Pro Glu Ser Ser 125 130 Leu Val Lys Ala Met Gly Asn Cys Ala 135140 83 amino acids amino acid unknown linear peptide 44 Pro Val Lys AspGlu Arg Glu Leu Lys Arg Gln Lys 1 5 10 Arg Lys Gln Ser Asn Arg Glu SerAla Arg Arg Ser 15 20 Arg Leu Arg Asn Glu Ala Glu Cys Glu Gln Thr Gln 2530 35 Gln Lys Val Leu Glu Leu Thr Ser Asp Asn Asp Arg 40 45 Leu Arg LysArg Val Glu Gln Leu Ser Arg Glu Leu 50 55 60 Asp Thr Leu Arg Gly Ile PheArg Gln Leu Pro Glu 65 70 Ser Ser Leu Val Lys Ala Met Gly Asn Cys Ala 7580 102 amino acids amino acid unknown linear peptide 45 Ala Ser Met ThrGly Gly Gln Gln Met Gly Arg Asp 1 5 10 Pro Leu Glu Glu Lys Val Phe ValPro Asp Glu Gln 15 20 Lys Asp Glu Lys Tyr Trp Thr Arg Arg Lys Lys Asn 2530 35 Asn Val Ala Ala Lys Arg Ser Arg Asp Ala Arg Arg 40 45 Leu Lys GluAsn Gln Thr Gln Gln Lys Val Leu Glu 50 55 60 Leu Thr Ser Asp Asn Asp ArgLeu Arg Lys Arg Val 65 70 Glu Gln Leu Ser Arg Glu Leu Asp Thr Leu ArgGly 75 80 Ile Phe Arg Gln Leu Pro Glu Ser Ser Leu Val Lys 85 90 95 AlaMet Gly Asn Cys Ala 100 28 base pairs nucleic acid unknown linear DNA(genomic) 46 GTCAGTCAGA TTGCGCAATA TCGGTCAG 28 10 base pairs nucleicacid unknown linear DNA (genomic) 47 ATTGCGCAAT 10 13 amino acids aminoacid unknown linear peptide 48 Met Asp Tyr Lys Asp Asp Asp Asp Lys LysLys Arg Lys 1 5 10 9 amino acids amino acid unknown linear peptide 49Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 8 amino acids amino acid unknownlinear peptide 50 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 11 amino acidsamino acid unknown linear peptide 51 Ala Ser Met Thr Gly Gly Gln Gln MetGly Arg 1 5 10 86 amino acids amino acid unknown linear peptide 52 MetAla Ser Met Thr Gly Gly Gln Gln Met Gly Arg 1 5 10 Asp Pro Asp Leu GluGln Arg Ala Glu Glu Leu Ala 15 20 Arg Glu Asn Glu Glu Leu Glu Lys GluAla Glu Glu 25 30 35 Leu Glu Gln Glu Asn Ala Glu Leu Glu Ala Glu Thr 4045 Asp Gln Leu Glu Asp Glu Lys Ser Ala Leu Gln Thr 50 55 60 Glu Ile AlaAsn Leu Leu Lys Glu Lys Glu Lys Leu 65 70 Glu Phe Ile Leu Ala Ala HisArg Pro Ala Cys Lys 75 80 Ile Pro 85 34 amino acids amino acid unknownlinear peptide 53 Asp Pro Asp Leu Glu Gln Arg Ala Glu Glu Leu Ala 1 5 10Arg Glu Asn Glu Glu Leu Glu Lys Glu Ala Glu Glu 15 20 Leu Glu Gln GluAsn Ala Glu Leu Glu Gln 25 30 34 amino acids amino acid unknown linearpeptide 54 Asp Pro Asp Leu Glu Gln Arg Ala Glu Glu Leu Ala 1 5 10 ArgGlu Asn Glu Glu Leu Glu Lys Glu Ala Glu Glu 15 20 Leu Glu Gln Glu LeuAla Glu Leu Glu Gln 25 30 31 amino acids amino acid unknown linearpeptide 55 Arg Ile Lys Ala Glu Arg Lys Arg Met Arg Asn Arg 1 5 10 IleAla Ala Ser Lys Cys Arg Lys Arg Lys Leu Glu 15 20 Arg Ile Ala Arg LeuGlu Glu 25 30 31 amino acids amino acid unknown linear peptide 56 SerAsn Glu Tyr Arg Val Arg Arg Glu Arg Asn Asn 1 5 10 Ile Ala Val Arg LysSer Arg Asp Lys Ala Lys Gln 15 20 Arg Asn Val Glu Thr Gln Gln 25 30 34amino acids amino acid unknown linear peptide 57 Gly Gly Gly Leu Glu GlnArg Ala Glu Glu Leu Ala 1 5 10 Arg Glu Asn Glu Glu Leu Glu Lys Glu AlaGlu Glu 15 20 Leu Glu Gln Glu Asn Ala Glu Leu Glu Gln 25 30 31 aminoacids amino acid unknown linear peptide 58 Ser Asn Glu Tyr Arg Val ArgArg Glu Arg Asn Asn 1 5 10 Ile Ala Val Arg Lys Ser Arg Asp Lys Ala LysGln 15 20 Arg Asn Val Glu Thr Gln Gln 25 30 31 amino acids amino acidunknown linear peptide 59 Asp Glu Lys Tyr Trp Thr Arg Arg Lys Lys AsnAsn 1 5 10 Val Ala Ala Lys Arg Ser Arg Asp Ala Arg Arg Leu 15 20 Lys GluAsn Gln Thr Gln Gln 25 30 31 amino acids amino acid unknown linearpeptide 60 Glu Leu Lys Arg Gln Lys Arg Lys Gln Ser Asn Arg 1 5 10 GluSer Ala Arg Arg Ser Arg Leu Arg Lys Gln Ala 15 20 Glu Cys Glu Gln LeuGln Gln 25 30 5 amino acids amino acid unknown linear peptide 61 Asp ProGly Gly Gly 1 5 4 amino acids amino acid unknown linear peptide 62 AspPro Glu Glu 1 6 amino acids amino acid unknown linear peptide 63 Asp ProAsp Glu Glu Glu 1 5 89 amino acids amino acid unknown linear peptide 64Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 1 5 10 Asp Pro Gly GlyGly Leu Glu Gln Arg Ala Glu Glu 15 20 Leu Ala Arg Glu Asn Glu Glu LeuGlu Lys Glu Ala 25 30 35 Glu Glu Leu Glu Gln Glu Asn Ala Glu Leu Glu Gln40 45 Glu Val Leu Glu Leu Glu Ser Arg Asn Asp Arg Leu 50 55 60 Arg LysGlu Val Glu Gln Leu Glu Arg Glu Leu Asp 65 70 Thr Leu Arg Gly Ile PheArg Gln Leu Pro Glu Ser 75 80 Ser Leu Val Lys Ala 85

What is claimed is:
 1. An isolated and purified nucleic acid bindingprotein having appended amino-terminally thereto an extension of aminoacid residues comprising a plurality of acidic amino acid residues. 2.The protein according to claim 1, wherein said protein is a DNA bindingprotein.
 3. The protein according to claim 1, wherein said protein is anRNA binding protein.
 4. The protein according to claim 1, wherein saidacidic amino acid residues dimerize with the basic region of a cellularDNA binding protein to inhibit the binding of said protein to DNA. 5.The protein according to claim 1, wherein said acidic amino acidresidues dimerize with the basic region of a cellular RNA bindingprotein to inhibit the binding of said protein to RNA.
 6. The proteinaccording to claim 4, said protein being a dominant negative to anaturally occurring cellular protein.
 7. The protein according to claim2, wherein said protein is a bZIP protein.
 8. The protein according toclaim 7, wherein said bZIP protein is selected from the group consistingof Fos, Jun, GCN4, VBP, GBF, opaque, CREB, C/EBP, PAR, ATF2 and plantG-box protein.
 9. The protein according to claim 2, wherein said proteinis a bHLH protein.
 10. The protein according to claim 8, wherein saidbHLH protein is selected from the group consisting of Myc, Max, and Mad.11. The protein according to claims 1, 2, or 3, wherein the acidic aminoacid residues are glutamic acid or aspartic acid.
 12. The proteinaccording to claim 1, 2, or 3, wherein said acidic extension comprisesfrom two to one-hundred amino acid residues.
 13. The protein accordingto claim 12, wherein said acidic extension comprises from three to fiftyamino acid residues.
 14. The protein according to claim 13, wherein saidacidic extension comprises from four to thirty amino acid residues. 15.The protein according to claim 14, wherein said acidic extensioncomprises twenty-eight amino acid residues.
 16. An isolated DNA moleculeconsisting essentially of the sequence as shown in SEQ ID NOS:1-52. 17.An isolated DNA molecule encoding a nucleic acid binding protein havingappended N-terminally thereto a plurality of acidic amino acid residues.18. A plasmid vector construct comprising the DNA molecule according toclaim 16 or claim 17, a promoter, a transcription initiation site, atranscription termination site, an origin of replication site, and apolyadenylation site, for expression in eukaryotic cells.
 19. The vectoraccording to claim 18, wherein said eukaryotic cells are selected fromthe group consisting of plant cells, yeast cells, and mammalian cells.20. A plasmid vector construct comprising the DNA molecule according toclaim 16 or claim 17, a promoter, a transcription initiation site and atranscription termination site, for expression in prokaryotic cells. 21.The construct according to claim 18, wherein said promoter is tissuespecific.
 22. The DNA molecule according to claim 16 or 17, wherein saidnucleic acid binding protein is a DNA binding protein.
 23. A method forproducing a dominant negative nucleic acid binding protein forinhibiting cell growth and proliferation, comprising: (a) preparing asequence of amino acids, wherein at least one amino acid of the sequenceis acidic to produce an acidic amino acid extension; and (b) appendingsaid acidic extension to the N-terminus of a multimerization or adimerization domain of said nucleic acid binding protein to create saiddominant negative protein.
 24. The method according to claim 23, whereinsaid dominant negative protein is a DNA binding protein.
 25. The methodaccording to claim 23, wherein said acidic extension comprises from twoto one-hundred amino acids.
 26. The method according to claim 25,wherein said acidic extension comprises from three to fifty amino acids.27. The method according to claim 26, wherein said acidic extensioncomprises from four to thirty amino acids.
 28. A method of controllingcell growth by inhibiting the function of a naturally occurring cellularprotein, comprising: (a) introducing into a cell the construct accordingto claim 18 under conditions allowing for the expression of saidacidically extended nucleic acid binding protein; (b) inhibiting thebinding of a cognate naturally occurring cellular nucleic acid bindingprotein to its target nucleic acid sequence by multimeric or dimericcomplexation between said expressed acidically extended nucleic acidbinding protein and said naturally occurring cellular protein.